Novel scavenger receptor

ABSTRACT

Novel scavenger receptors having an SR structure and a collectin-like structure are provided, which can be utilized in the elucidation of mechanisms of macrophage and basic immunity; in the elucidation of mechanisms of the development of a wide variety of diseases such as arteriosclerosis, diabetic complications and Alzheimer&#39;s disease, hyper β-lipoproteinemia, hypercholesterolemia, hypertriglyceridemia, hypo α-lipoproteinemia, transplantation, atherectomy, post angiogenic restenosis, bacterial infections; in the diagnostic, prophylactic and therapeutic methods thereof; and in the development of reagents and drugs for the same. The novel scavenger receptors include proteins comprising an amino acid sequence set out in SEQ ID NO: 2, 4 or 24 or proteins having equivalent properties to the same, or derivatives or fragments thereof as well as isolated polynucleotides comprising a nucleotide sequence encoding these proteins, and related molecules such as antibodies, antagonists and the like. Also disclosed are methods for the treatment using the same.

This application is a divisional of U.S. patent application Ser. No.10/203,860, filed Sep. 30, 2002, which is the U.S. national phase ofInternational Application No. PCT/JP01/00874, filed Feb. 8, 2001, whichclaims priority benefit of Japanese Patent Application No. JP2000-35155, filed Feb. 14, 2000, and Japanese Patent Application No. JP2000-309068, filed Oct. 10, 2000. All priority applications are herebyincorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to isolated human and mouse novelscavenger receptors (herein referred to as “hSRCL-P1” and “mSRCL-P1”respectively, or merely as “SRCL-P1” when discrimination is notintended), genes and proteins, the homologues, mutants, modified formsand polymorphic variants thereof (these are collectively referred to as“derivatives”), fragments thereof (hereinafter collectively referred toas “SRCL-P1s” for all of these), and the detection thereof. The presentinvention further relates to compositions which comprise SRCL-P1s forpharmaceutical use, diagnostic use and research use, and methods for theproduction and use of the same. Additionally, the present inventionrelates to agonists and antagonists of SRCL-P1s proteins, as well asmethods for screening drugs using SRCL-P1s. Moreover, the presentinvention relates to expression vectors comprising SRCL-P1s gene,transformed cells that were transformed with the expression vector,antibodies to SRCL-P1 protein, and cells that produce the antibody.

BACKGROUND OF THE INVENTION

Pathological features in lesions at an early stage of atherosclerosisinvolve the event of increase of foam cells in artery walls. Scavengerreceptors (hereinafter abbreviated as “SR”) that are present on a cellmembrane of a macrophage (Krieger, M. et al., Annu. Rev. Biochem., 63,601-637, 1994) lack negative feed back regulation by cholesterol, alienfrom LDL receptors. Thus, the receptor itself changes into foam cellsthrough actively incorporatings modified LDL (low density lipoproteinthat is a complex of cholesterol and a lipoprotein) to accumulatebeneath the vascular endothelial cells. Therefore, macrophages and SRsthereof have been believed to play important roles in the establishmentof pathosis of atherosclerosis (Brown, M. S. et al., Nature, 343,508-509, 1990; Kurihara, Y. A. et al., Current Opinion in Lipidology, 2,295-300, 1991; Krieger, M., TIBS, 17, 141-146, 1992; Krieger, M. et al.,J. Biol. Chem., 268(7), 4569-4572, 1993).

Continuous hyperglycemia in a living body resulting from diabetes causesnonenzymatic glycation of various proteins, thereby leading theproduction of Maillard reaction-advanced end products (AGE: advancedglycation end products), which are final products in a glycation processvia a Schiff base and an Amadori compound. AGE having an injuriousaction on cells adversely affects through the binding to macrophages,vascular endothelial cells, hepatic cells, renal mesangium cells and thelike via AGE receptors. For example, it is known that secretion ofcytokines such as TNF (Tumor Necrosis Factor), IL-1 (Interleukine-1) andplatelet derived growth factor (PDGF) is accelerated upon binding of AGEto a macrophage, thereby causing cell injuries characteristic todiabetic complications. SR is believed to participate profoundly indiabetic complications such as diabetic nephropathy, diabeticretinopathy, diabetic neuropathy, on the basis of the findings that SRis one of the receptors involving in incorporation and degradation ofAGE (Araki, N. et al., Eur. J. Biochem., 230, 408-415, 1995; Suzuki, H.et al., Circulation, 92, 1-428, 1995), and that degradative activity ofAGE is lowered to a level of third in an SR-double knockout mouse.Further, when an excessive AGE albumin is administered to a rat, AGE wasfound to deposit in kidney, thereby developing and glomerulosclerosis(Vlassara, H. et al., Proc. Natl. Acad. Sci. USA, 91, 11704-11708,1994). Accordingly, SR, which recognizes AGE, is anticipated toprofoundly involve in glomerulosclerosis.

In addition, SR is believed to involve in Alzheimer's disease.Pathological features of Alzheimer's disease concern senile plaques thatare deposits of β-amyloid. β-amyloid has been reported to activatemicroglia cells via SRs that are expressed on the microglia cells togenerate active oxygen, leading to the expression of neurotoxicity(Nature, 382, 716-719, 1996).

Examples of ligand for SRs include: ligands having negative charge,e.g., modified LDL such as acetylated LDL (AcLDL), oxidized LDL (OxLDL)and the like, modified proteins such as maleylated BSA and the like,quadruple helical nucleic acids such as polyinosinic acids and the like,polysaccharides such as dextran sulfate and fucoidane and the like,acidic phospholipids such as phosphatidylserine, phosphatidylinositoland the like, endotoxin (LPS), AGE, senile cells apoptotic cells, andthe like, although differences in specificity thereof may existdepending on the differences of molecular species of SRs. Additionally,SR is believed to play an important role in removal of foreignsubstances, metabolic decomposition products and the like, because SRextensively recognizes various modified substances and a wide variety offoreign substances such as viruses in a living body (Hampton, R. Y. etal., Nature, 352, 342-344, 1991; Tokuda, H. et al., Biochem. Biophys.Res. Commun., 196(1), 8-24, 1993; Pearson, A. M. et al., J. Biol. Chem.,268, 3546-3554, 1993; Dunne, D. W. et al., Proc. Natl. Acad. Sci. USA,91, 1863-1867, 1994; Freeman, M. W.; Current Opinion in Lipidology, 5,143-148, 1994).

SRs have been expressed in hepatic sinusoidal endothelial cells (Eskild,W. et al., Elsevier Biomedical N.Y., 255-262, 1982), vascularendothelial cells (Baker, D. P. et al., Arteriosclerosis, 4, 248-255,1984; Bickel, P. E. et al., J. Clin. Invest., 90, 1450-1457, 1992),blood smooth muscle cells (Pitas, R. E. et al., J. Biol. Chem., 265,12722-12727, 1990; Bickel, P. E. et al., J. Clin. Invest., 90,1450-1457, 1992), fibroblasts (Pitas, R. E. et al., J. Biol. Chem., 265,12722-12727, 1990), and the like as well as in macrophages. Further, SRshave been classified into SRA, SRB, SRC (Peason, A. et al., Proc. Natl.Acad. Sci. USA, 92, 4056-4060, 1995), FcγRIIB2 (Stanton, L. W. et al.,J. Biol. Chem., 270, 22446-22451, 1992) and macrosialin (CD68)(Ramprasad, M. P. et al., Proc. Natl. Acad. Sci. USA, 92, 9580-9584,1995), human vascular endothelial OxLDL receptor (LOX-1□lectin-likeoxidized LDL receptor) (Sawamura, T. et al., Nature, 386, 73, 1997).Moreover, SRA has been classified into SR-AI and SR-AII (Kodama, T. etal., Nature, 343, 531-535, 1990), and MARCO (a novel macrophage receptorwith collagenous structure) (Elomaa, O. et al., Cell, 80, 603-609,1995); SRB has been classified into CD36 (Endemann, G. et al., J. Biol.Chem., 268, 11811-11816, 1993) and SR-BI (Acton, S. L. et al., J. Biol.Chem., 269, 21003-21009, 1994).

SR-AI and SR-AII are homotrimers, which are of inside-out typetransmembrane proteins of which N-terminus resides within the cell. Theprotein is structurally revealed to have several domains such as acollagen-like domain, α-helical coiled coil domain and a cysteine-richdomain, and the like in its extracellular portion (Rohrer, L. et al.,Nature, 343, 570, 1990; Matsumoto, A. et al., Proc. Natl. Acad. Sci.USA, 87, 9133, 1990). The collagen-like domain has a structurecharacteristic in collagen, (Gly-Xaa-Yaa)n, wherein Xaa and Yaa may beany one of amino acid residues, and this domain functions as aligand-binding site. The α-helical coiled coil domain is a dexiotropichepted repeat which turns two times at every seven amino acids, namelyhaving a structure of α-helical coiled coil. The three polypeptides forma homotrimer with hydrophobic amino acids such as leucine and isoleucinethat are present at every seven amino acids being directed to inside ofthe molecule, whilst having polar amino acids and carbohydratechain-binding site outside thereof (leucine zipper). Roles of thedomains involve retention of the homotrimer structure, as well asbinding to the ligands such as modified LDL to incorporate them into thecells, and changing a tertiary structure of the receptor depending ondecrease of pH in endosome, finally resulting in the dissociation of theligands.

Intracellular domain of the protein has a tight turn structure, which ischaracteristically found in an endocytotic signal, similarly to thestructures including NPXY sequence found in LDL receptors or insulinreceptors and YXRF sequence found in transferrin receptors. It issuggested that endocytosis may be suppressed when these sequences aredeleted.

SR-A1 and SR-A2 arise from alternative splicing of mRNA coding acysteine-rich domain. SR-AI has 110 amino acids corresponding to thedomain, whilst SR-AII has corresponding 17 amino acids. SR-AI and SR-AIIare expressed in at least peripheral macrophage derived from monocyte,pulmonary alveolus macrophage and hepatic Kupffer cell. It is revealedthat they participate in a host defense system in a living body, forexample, arteriosclerosis, Calcium ion-independent cell adhesion and thelike (Krieger, M. et al., Annu. Rev. Biochem., 63, 601-637, 1994; Wada,Y et al., Ann. N.Y. Acad. Sci., 748, 226-239, 1995; Fraser, I. P. etal., Nature, 364, 343, 1993). Further, OxLDL is present withinmacrophages of arteriosclerosis foci. In addition, SR-AI and SR-AII areabundantly expressed on the cell membrane of macrophage, and theelevation of blood lipoprotein by lipid absorption is suppressed in atransgenic mouse for SR-AI. Accordingly, it is envisaged that SR-AI andSR-AII play important roles in incorporation of Ox LDL.

To the contrary, although MACRO classified into SRA has a similarstructure as that of SR-Al, it has no α-helical coiled coil domain,which is characterized by having a long collagen-like domain. MACRO isexpressed in spleen macrophage, lymph node macrophage and the like,which is believed to function in a host defense mechanism againstbacterial infection in a living body taking into account of thespecificity of the ligands thereof.

Suzuki et al., successfully produced an SRA-knockout mouse through thesubstitution of the fourth exon that is a common part between SR-AI andSR-AII with a neomycin resistant gene (Suzuki, H. et al., Nature, 386,292-296, 1997). Immune disorder has been observed in the SRA-knock outmouse in comparison with the wild type, and exhibits a high rate ofinfection with Listeria and herpes simplex virus. In addition, it isindicated that SRA participates in phagocytosis of T cells havingapoptosis occurred, and that the phagocytic capacity is reduced in theSRA-knockout mouse in comparison with the wild type (Platt, N. et al.,Proc. Natl. Acad. Sci. USA, 93, 12456, 1996). Furthermore, in a doubleknockout mouse obtained by the mating of the SRA-knock out mouse and anapoE deficient mouse (Plump, A. S. et al., Cell, 71, 343, 1992; Zhag, S.H. et al., J. Clin. Invest., 94, 937, 1994) that is an animal model forarteriosclerosis, it is indicated that the area of arteriosclerosis fociis significantly smaller than that of the apoE deficient mouse (Suzuki,H. et al., Nature, 386, 292-296, 1997).

Thus, SR can be utilized in the elucidation of functions of macrophage,the elucidation of mechanisms of development of various types ofdiseases including for example, arteriosclerosis, diabetic complicationsand AD, hyper β-lipoproteinemia, hypercholesterolemia,hypertriglyceridemia, hypo α-lipoproteinemia, transplantation,atherectomy, post angiogenic restenosis and the like, as well asdiagnostic, prophylactic, therapeutic methods thereof, and in thedevelopment of reagents and drugs for the same. Accordingly, to findnovel molecular species belonging to this family can be the means tosolve the above-described problem to be solved.

Besides, a complement system that plays an important role in a hostdefense mechanism is known to include: a classical pathway in which animmunoglobulin serves as a recognition molecule followed by theactivation of C1 that is the first component of the complement; and analternative pathway in which C3, which is the third component of thecomplement, is directly coupled to foreign substances such as bacteria.In addition to these pathways of the complement activation, a lectinpathway was illustrated in which a mannose binding protein (hereinafterreferred to as “MBP”), which is a serum lectin, activates the complementsystem through the direct recognition of and coupling with acarbohydrate chain on the surface of the foreign substance, in recentyears (Sato, T. et al., Int. Immunol., 6, 665-669, 1994).

MBP is a C type lectin which specifically binds to mannose,N-acetylglucosamine and the like in the presence of Calcium ion, ofwhich structure comprises a collagen-like domain containing at least(Gly-Xaa-Yaa)_(n), and carbohydrate recognition domain (CRD). Similarlyto MBP, lectins having a collagen-like domain and CRD are genericallycalled as collectin (Malhotora, R. et al., Eur. J. Immunol., 22,1437-1445, 1992), which include collectin-43 (CL-43), surfactant proteinA (SP-A), surfactant protein D (SP-D), bovine conglutinin (BKg) and thelike, in addition to MBP. Collectin has an opsonic activity, which isbelieved to participate in basal immunity against a variety ofmicroorganisms such as bacteria and viruses (Kawasaki, N. et al., J.Biochem., 106, 483-489, 1989; Ikeda, K. et al., J. Biol. Chem., 262,7451-7454, 1987; Ohta, M. et al., J. Biol. Chem., 265, 1980-1984, 1990;Summerfield, J. A. et al., Lancet, 345, 886, 1995).

These collectins are known to constitute from a basic structurecontaining characteristic domains such as (1) CRD and (2) collagen-likedomain and the like as shown in FIG. 1(a) (Malhortra et al., Eur. J.Immunol., 22, 1437-1445, 1992). This basic structure forms a subunitthrough composing a triple helix at the collagen-like domain, and thusthese subunits further form an oligomer structure such as trimer,tetramer, hexamer and the like.

Recently, collectins were suggested to participate in non-specificimmune response, e.g., it was reported that for example, they areplaying important roles in neutralizing and excluding variousmicroorganisms in infants having maternal antibodies from the mother ornonspecific defense systems which were insufficiently developed (Superet al., Lancet, 2, 1236-1239, 1989). Moreover, results of investigationare reported involving in roles of these collectins in the body defensesystem of a host, which for example, suggest that the host becomes moresusceptible to infections through the lowered concentration of MBP inblood resulting from genetic mutation of MBP (Sumiya et al., Lancet,337, 1569-1570, 1991). In addition, it was reported that serum MBPcontent shows a lowered level upon the failure of opsonization (Madsen,H. O. et al., Immuno genetics, 40, 37-44, 1994), whilst bacterialinfections readily occur (Garred, P. et al., Lancet, 346, 941-943,1995). Therefore, MBP may be believed to play important roles in animmune system.

The present inventors previously found that BKg and MBP inhibitinfections by H1 and H2 types influenzae A viruses as well as ahaemagglutination activity (Wakamiya et al., Glycoconjugate J., 8, 235,1991; Wakamiya et al., Biochem. Biophys. Res. Comm., 187, 1270-1278,1992). Thereafter, a cDNA clone encoding BKg was obtained, and therelevance between BKg and SP-D and the like has been also found (Suzukiet al., Biochem. Biophys. Res. Comm., 191, 335-342, 1993).

Likewise, collectins are substances to which usefulness in theelucidation of host defense mechanism and utilities as a biologicallyactive substance are expected. Thus, the finding of novel molecularspecies belonging to this family may greatly contribute in variousmedical fields and biological fields in addition to the therapy ofinfectious diseases.

DISCLOSURE OF THE INVENTION

The object of the present invention is to provide a novel scavengerreceptor that can be utilized in the elucidation of mechanisms ofmacrophage and basic immunity; in the elucidation of mechanisms of thedevelopment of a wide variety of diseases such as arteriosclerosis,diabetic complications and Alzheimer's disease, hyper β-lipoproteinemia,hypercholesterolemia, hypertriglyceridemia, hypo α-lipoproteinemia,transplantation, atherectomy, post angiogenic restenosis, bacterialinfections; in the diagnostic, prophylactic and therapeutic methodsthereof; and for the development of reagents and drugs for the same.

Accordingly, the aspects provided by the present invention are asdescribed below.

(1) A protein comprising an amino acid sequence consisting of 742 aminoacids set out in amino acid position 1 to 742 of SEQ ID NO: 2, or aprotein comprising an amino acid sequence set out in SEQ ID NO: 2 havingdeletion, substitution or addition of one or several amino acids thereinand having an equal property to that of the protein comprising an aminoacid sequence set out in amino acid position 1 to 742 of SEQ ID NO: 2,or a derivative or a fragment thereof;

(2) An isolated polynucleotide comprising a nucleotide sequence set outin nucleotide position 74 to 2299 of SEQ ID NO: 1, a nucleotide sequenceencoding an amino acid sequence set out in amino acid position 1 to 742of SEQ ID NO: 2 or a fragment thereof, or a nucleotide sequence thathybridizes to any one of said nucleotide sequences or nucleotidesequences complementary thereto under a stringent condition and encodesa protein having an equal property to that of the protein comprising anamino acid sequence set out in amino acid position 1 to 742 of SEQ IDNO: 2;

(3) A protein comprising an amino acid sequence set out in amino acidposition 1 to 618 of SEQ ID NO: 24, or a protein comprising an aminoacid sequence set out in SEQ ID NO: 24 having deletion, substitution oraddition of one or several amino acids therein and having an equalproperty to that of the protein comprising an amino acid sequence setout in amino acid position 1 to 618 of SEQ ID NO: 24, or a derivative ora fragment thereof;

(4) An isolated polynucleotide comprising a nucleotide sequence set outin nucleotide position 74 to 1933 of SEQ ID NO: 23, a nucleotidesequence encoding an amino acid sequence set out in amino acid position1 to 618 of SEQ ID NO: 24 or a fragment thereof, or a nucleotidesequence that hybridizes to any one of said nucleotide sequences ornucleotide sequences complementary thereto under a stringent conditionand encodes a protein having an equal property to that of the proteincomprising an amino acid sequence set out in amino acid position 1 to618 of SEQ ID NO: 24;

(5) A protein comprising an amino acid sequence consisting of 742 aminoacids set out in amino acid position 1 to 742 of SEQ ID NO: 4, or aprotein comprising an amino acid sequence set out in SEQ ID NO: 2 havingdeletion, substitution or addition of one or several amino acids thereinand having an equal property to that of the protein comprising an aminoacid sequence set out in amino acid position 1 to 742 of SEQ ID NO: 2,or a derivative or a fragment thereof;

(6) An isolated polynucleotide comprising a nucleotide sequence set outin nucleotide position 74 to 2299 of SEQ ID NO: 3, a nucleotide sequenceencoding an amino acid sequence set out in amino acid position 1 to 742of SEQ ID NO: 2 or a fragment thereof, or a nucleotide sequence thathybridizes to any one of said nucleotide sequences or nucleotidesequences complementary thereto under a stringent condition and encodesa protein having an equal property to that of the protein comprising anamino acid sequence set out in amino acid position 1 to 742 of SEQ IDNO: 2;

(7) A vector comprising a polynucleotide according to (2), (4) or (6);

(8) A transformed cell carrying a polynucleotide according to (2), (4)or (6) in a manner to allow the expression;

(9) A method for the production of a protein which comprises the step ofculturing a cell transformed with the polynucleotide according to (2) or(4), and collecting thus produced hSRCL-P1 protein;

(10) A method for the production of a protein which comprises the stepsof culturing a cell transformed with the polynucleotide according to(6), and collecting thus produced mSRCL-P1 protein;

(11) The method according to (9) or (10) wherein said cell isEscherichia coli, an animal cell or an insect cell;

(12) A transgenic non-human animal having an altered expression level ofSRCL-P1 gene;

(13) The transgenic non-human animal according to (12) wherein saidSRCL-P1 gene is cDNA, genomic DNA or synthesized DNA encoding SRCL-P1;

(14) The transgenic non-human animal according to (13) wherein theexpression level is altered by causing the mutation at a gene expressionregulatory site;

(15) A knockout mouse wherein a function of mSRCL-P1 gene is deficient;

(16) An antibody to the protein according to (1), (3) or (5), or afragment thereof;

(17) The antibody according to (16), which is a polyclonal antibody, amonoclonal antibody or a peptide antibody.

(18) A method for the production of a monoclonal antibody to the proteinor the fragment thereof according to (1), (3) or (5) which comprisesadministering the protein or a fragment thereof according to (1), (3) or(5) to a warm-blooded animal other than human, selecting the animal thatexhibits an antibody titer, collecting a spleen or a lymph node from theanimal, fusing antibody-producing cells contained therein with myelomacells to prepare a hybridoma that produces a monoclonal antibody;

(19) A method for quantitatively determining an SRCL-P1 protein or afragment thereof on the basis of an immunological binding between theantibody according to (16) or (17) and the SRCL-P1 protein or a fragmentthereof;

(20) A method for detecting an SRCL-P1 protein or a fragment thereof onthe basis of an immunological binding between the antibody according to(16) or (17) and the SRCL-P1 protein or a fragment thereof;

(21) An agonist that stimulates an activity of the protein according to(1), (3) or (5);

(22) An antagonist that inhibits an activity or the activation of theprotein according to (1), (3) or (5);

(23) A method for screening a drug wherein the protein according to (1),(3) or (5) is used;

(24) A drug which is obtained by the method for the screening accordingto (23);

(25) A method for screening a drug for the treatment of a pathologicalstate involved in the accumulation of oxidized LDL, which comprises thestep of identifying a candidate drug for the treatment of a pathologicalstate involved in the accumulation of oxidized LDL by an inhibitoryability of the candidate drug toward the binding between the proteinaccording to (1), (3) or (5) and oxidized LDL, which is evaluated bycomparing the amount of binding between the protein and oxidized LDL inthe presence and absence of the candidate drug;

(26) A drug obtained by the method for the screening according to (25);

(27) A method for the treatment of a pathological state involved in theaccumulation of oxidized LDL, which comprises the step of inhibiting thebinding between an SRCL-P1 protein or a fragment thereof and oxidizedLDL using the drug according to (26);

(28) A pharmaceutical composition for the treatment of a pathologicalstate involved in the accumulation of oxidized LDL comprising the drugaccording to (26);

(29) A method for screening a drug for the treatment of a pathologicalstate involved in the binding of AGE to cells, which comprises the stepof identifying a candidate drug for the treatment of a pathologicalstate involved in the binding of AGE to cells by an inhibitory abilityof the candidate drug toward the binding between the protein accordingto (1), (3) or (5) and AGE, which is evaluated by comparing the amountof binding between the protein and AGE in the presence and absence ofthe candidate drug;

(30) A drug obtained by the method for the screening according to (29);

(31) A method for the treatment of a pathological state involved in thebinding of AGE to cells, which comprises the step of inhibiting thebinding between an SRCL-P1 protein or a fragment thereof and AGE usingthe drug according to (30); and

(32) A pharmaceutical composition for the treatment of a pathologicalstate involved in the binding of AGE to cells comprising the drugaccording to (30);

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing illustrating a basic structure ofprincipal collectins reported heretofore and an overview of the protein.

FIG. 2 is a drawing illustrating a preceding half of an alignment ofamino acid sequences of three kinds of collectins (Human MBP, SEQ Id NO:29; Human SP-A, SEQ ID NO: 30; and Human SP-D, SEQ ID NO: 31) reportedheretofore.

FIG. 3 is a drawing illustrating the latter half of the alignment setforth in FIG. 2 of collectins Human MBP, SEQ ID NO: 29; Human SP-A, SEQID NO: 30 and Human SP-D, SEQ ID NO: 31.

FIG. 4 (b) depicts drawings showing a nucleotide sequence read by asequencer and each primer employed for the sequencing of the novelscavenger receptor of the present invention; and FIG. 4 (a) depicts adrawing showing ORF of the novel collectin obtained.

FIG. 5 is a drawing illustrating a manner how A: yeast, B: gram negativebacteria (Escherichia coli), and C: gram positive bacteria(Staphylococcus aureus) specifically bind to cells that are expressinghSRCL-P1.

FIG. 6 is a drawing illustrating a manner how A: oxidized LDL, B:mannose, and C: AGE specifically bind to cells that are expressinghSRCL-P1.

FIG. 7 is a drawing illustrating a manner how yeast is incorporated intocells that are expressing hSRCL-P1.

FIG. 8 is a drawing illustrating a manner how hSRCL-P1 is expressed inA: healthy human heart vascular endothelial cells, and B: mouse heartvascular endothelial cells.

BEST EMBODIMENT FOR CARRYING OUT THE INVENTION

The present inventors successfully cloned human and mouse novel SR. Acollectin domain containing CRD is present at C-terminuses of the novelSR (SRCL-P1), which is believed to participate in basic immunity, andthe entire structure of the novel SR was similar to that of SRA, inparticular, SR-AI. More specifically, it was constituted at least from atransmembrane domain comprising a leucine zipper that has leucine unitsrepeated four times, an α-helical coiled coil domain, a collagen-likedomain, a neck domain, a CRD domain, starting from the N-terminuses.Three molecules having such a characteristic structure are envisaged toform a homotrimer through the formation of an α-helix at the coiled coildomain and the formation of a triple helix at the collagen-like domain.Further, the collagen-like domain is speculated to be positively chargedunder a condition of physiological pH. In addition, SRCL-P1 protein hadnumerous carbohydrate chain binding sites

The hSRCL-P1 gene and mSRCL-P1 gene used herein include polynucleotidescomprising a nucleotide sequence set out in SEQ ID NO: 1 or 3,derivatives thereof (homologues, mutants, modified forms and polymorphicvariants), and fragments thereof, unless otherwise stated. Further, thehSRCL-P1 protein and mSRCL-P1 protein used herein comprise amino acidsequence set out in SEQ ID NO: 2 or 4, derivatives thereof and fragmentsthereof, unless otherwise stated. These may be derived from naturalsources, or artificially synthesized. The present invention includeswhole of the substances as described above.

Examples of hSRCL-P1 include proteins comprising amino acids set out inSEQ ID NO: 24 (a mutant of the protein set out in SEQ ID NO: 1 withdeletion of a part of the collagen-like domain and the neck domain,i.e., amino acid residues of position 483 to 606), and examples ofmutants of the polynucleotide set out in SEQ ID NO: 1 include apolynucleotide set out in SEQ ID NO: 23 which encodes the protein setout in SEQ ID NO: 24.

Moreover, the present invention also involves amino acid sequencessubstantially similar to the amino acid sequence set out in SEQ ID NO: 2or 4, and nucleotide sequences encoding amino acid sequencessubstantially similar to the amino acid sequence set out in SEQ ID NO: 2or 4. Furthermore, proteins comprising these amino acid sequences arealso involved. The amino acid sequence substantially similar to theamino acid sequence set out in SEQ ID NO: 2 or 4 refers to the aminoacid sequence having alteration such as substitution, deletion, additionand/or insertion of one or several amino acids therein as long as theprotein has an equal property to those of the protein comprising anamino acid sequence set out in SEQ ID NO: 2 or 4, that is to say;activity, function and tertiary structure due to the structure whichcomprises a transmembrane domain containing a leucine zipper structure,and α-helical coiled coil domain and collagen-like domain, which arecharacteristic in SR. These may be derived from natural sources, orartificially synthesized.

Furthermore, the present invention also involves a nucleotide sequenceset out in SEQ ID NO: 1 or 3 or a nucleotide sequence comprising afragment thereof, or a nucleotide sequence that can hybridize to anucleotide sequence complementary thereto (hereinafter, referred to as“specified sequence”) under a stringent condition. The stringentcondition according to the present invention may involve a condition forexample; incubating in a solution containing 5×SSC, 5% Denhardt'ssolution (0.1% BSA, 0.1% Ficoll 400, 0.1% PVP), 0.5% SDS, and 20 μg/mlmodified sermon sperm DNA at 37° C. overnight followed by a wash with2×SSC containing 0.1% SDS at room temperature. SSPE may be employed inplace of SSC. Thus resultant nucleotide sequence is speculated toexhibit at least 50% or more homology with the specified sequence. Manyof the proteins encoded by the nucleotide sequence that hybridize to thespecified sequence under a stringent hybridization condition arebelieved to have an equal property to SRCL-P1 protein. Therefore, suchproteins are also involved in the present invention as long as they havean equal property to SRCL-P1 protein.

In particular, the amino acid sequence of hSRCL-P1 set out in SEQ ID NO:2 (amino acid position 1 to 742) represents a protein consisting of 742amino acids, and thus the nucleotide sequence encoding the same consistsof 2226 nucleotides. Characteristic amino acid sequences such as thoseof a leucine zipper domain, an α-helical coiled coil domain, acollagen-like domain, a neck domain, a CRD domain and the like werepresent in the sequence. That is to say, a leucine zipper domaindesignated by amino acid position 36-57, an α-helical coiled coil domaindesignated by amino acid position 72-426 (according to COILS Program) oramino acid position 81-431 (according to MultiCoil Program), acollagen-like domain designated by amino acid position 443-589, a neckdomain designated by amino acid position 590-606, a CRD domaindesignated by 607-742 and the like were present. Other domains includefor example, an extracellular domain designated by amino acid position63-742 (according to TMHMM1.0 program) or amino acid position 58-742(according to TMpred program), an intracellular domain designated byamino acid position 1-39 (according to TMHMM1.0 program) or amino acidposition 1-37 (according to TMpred program), a transmembrane domaindesignated by amino acid position 40-62 (according to TMHMM1.0 program)or amino acid position 38-57 (according to TMpred program), acollectin-like domain designated by amino acid position 443-742.Moreover, a C type lectin motif designated by amino acid position708-730 was also included. The nucleotide sequence encoding this proteinis set out in SEQ ID NO: 1.

The amino acid sequence of mSRCL-P1 (amino acid sequence 1-742) set outin SEQ ID NO: 4 represents a protein consisting of 742 amino acids, andthus the nucleotide sequence encoding the same consists of 2226 bases.Similarly to hSRCL-P1 set out in SEQ ID NO: 2, characteristic amino acidsequences such as a leucine zipper domain, an α-helical coiled coildomain, a collagen-like domain, a neck domain, a CRD domain, a C typelectin motif and the like were present in the sequence. The nucleotidesequence encoding this protein is set out in SEQ ID NO: 3.

Homologues used herein refer to nucleotide sequences or amino acidsequences that bear high homology, which are homologous at least 50% ormore, preferably 70% or more, more preferably 90% or more. When deletionor insertion is present in the sequence, homologous search may beconducted which allows for gap junction. For example, the search may beperformed using a procedure of multiple alignment (trade name: SODHO,Fujitsu Limited). In addition, as the algorithm for searching homology,Smith-Waterman algorithm, which is the most accurate, may be employed.Alternatively, FASTA or BLAST may be also utilized via the Internet.

Mutants used herein include for example, those resulting from allele,Single Nucleotide Polymorphism (SNP) and the like. Furthermore, thenucleotide sequence of the present invention may also include themutated nucleotide sequences derived from the changes in the range ofdegeneracy of the codon. Partial alteration of the codon of a nucleotidesequence may be achieved according to a routine method using such sitedirected mutagenesis methods as those in which a primer is employedconsisting of a synthesized oligonucleotide that encodes the desiredalteration (Mark, D. F. et al., Proc. Natl. Acad. Sci. USA., 81, 5662,1984). Thus resultant artificial genetic mutants are also involved inthe nucleotide sequence of the present invention. Furthermore, themutated amino acids translated by the mutated codons have preferablysimilar properties to those of the normal amino acid even in the casewhere the mutation is beyond the range of degeneracy of the codon. Themutation may preferably be as follows, which are among amino acidshaving similar properties, functions, characteristics and the like, forexample: the mutation among aliphatic amino acids such as alanine,valine, leucine and isoleucine; the mutation among neutral amino acidssuch as glycine, alanine, serine, threonine, valine, leucine,isoleucine, cysteine, methionine, phenylalanine, tyrosine, proline,tryptophan, asparagines and glutamine; the mutation among acidic aminoacids such as aspartic acid and glutamic acid; the mutation among basicamino acids such as arginine, lysine and histidine; the mutation amongserine and threonine, having a hydroxyl group; the mutation amongphenylalanine and tyrosine, having an aromatic ring; and the like. Theseartificially or naturally mutated proteins are also included in theprotein of the present invention. For the artificial mutation,site-directed mutagenesis may be caused using a PCR method, andalternatively, other known methods may be used to cause mutation at anyoptional site.

Modified forms used herein may be prepared using conventionaltechniques, for example, by acetylation, acylation, ADP-ribosylation,amidation, myristoylation, glycosylation, hydroxylation,phosphorylation, sulfation, formylation, methylation,polyethyleneglycolation, lipid coupling, nucleotide coupling, metalcoupling (calcium addition and the like), fusion with other protein(albumin and the like), dimerization, and the like. For example, sinceglycosylation does not occur in Escherichia coli as a host, theexpression may be conducted in eucaryotic cells when glycosylation isintended. Insect cells may be also used because glycosylation proceedspost-translationally, similarly to in mammalian cells.

Polymorphic variants used herein involve for example, polymorphismscaused by structural or conformational differences in chromosomal DNA,polymorphisms resulting from a change of a gene into its allelic gene,or the like. In general, genes of eucaryotic cells often exhibitpolymorphic event, and this event may lead to the substitution of one ormore amino acid(s) whilst the activity of the protein may be retained.Therefore, any of the genes encoding a protein obtained by artificiallymodifying the gene encoding any of the amino acid sequence set out inSEQ ID NO: 2 or 4 is involved in the present invention as far as theprotein has a characteristic function of a gene of the presentinvention. In addition, any of the proteins comprising artificiallymodified amino acid sequence set out in SEQ ID NO: 2 or 4 is involved inthe present invention as far as it has a property of a protein of thepresent invention. The modification is construed as involvingsubstitution, deletion, addition and/or insertion.

Fragments used herein refer to any optional fragments derived from theamino acid sequence of SRCL-P1 described above, which include forexample, an extracellular domain, an intracellular domain, atransmembrane domain, a leucine zipper domain, an α-helical coiled coildomain, a collagen-like domain, a neck domain, a CRD domain, acollectin-like domain, a hydrophobic domain (a neck domain, atransmembrane domain and the like), a hydrophilic domain (domains otherthan hydrophobic domains), and the like, as well as fragments obtainedby the fusion of these fragments. For example, in the amino acidsequence of hSRCL-P1 set out in SEQ ID NO: 2, the fragments included maybe: a fragment comprising amino acids of from position 58-63 to position742 that form a soluble receptor but lacks a transmembrane domain; afragment comprising amino acids of from position about 1 to 606 thatform a transmembrane scavenger receptor but lacks a CRD domain; afragment comprising amino acids of from position about 36 to position426-431 that form a soluble scavenger receptor containing a leucinezipper and an α-helical coiled coil domain; and a fragment comprisingamino acids of from position 1 to 589 which lacks a CRD domain and aneck domain.

Method for Obtaining SRCL-P1 Gene

A SRCL-P1 gene according to the present invention may be those obtainedthrough any methods. For example, the nucleotide sequence encodingSRCL-P1 of the present invention can be obtained by preparing mRNA fromthe cells that are expressing the protein, and altering it into a doublestranded DNA by a conventional technique. For the preparation of mRNA,guanidine isothiocyanate calcium chloride method (Chirwin, et al.,Biochemistry, 18, 5294, 1979) and the like can be employed. For thepreparation of poly(A)^(□) RNA from total RNA, supports bound witholigo(dT), for example, affinity chromatography in which sepharose orlatex particles are used, can be employed. Double stranded cDNA can beobtained by using thus obtained RNA described above as a template totreat with reverse transcriptase using oligo(dT) that is complementaryto poly(A) chain present at 3′-terminus, or a random primer or asynthesized oligonucleotide corresponding to a part of the amino acidsequence of SRCL-P1 as a primer (Mol. Cell. Biol., 2, 161, 1982; Mol.Cell. Biol., 3, 280, 1983; Gene, 25, 263, 1983); and treating thusresulting cDNA with for example, E. coli RNaseH, E. coli DNA polymerase1, E. coli DNA ligase to alter into the DNA chain. A cDNA library can beproduced by incorporating this cDNA into a plasmid vector, phage vector,cosmid vector to transform E. coli, or by transfecting it into E. colifollowing in vitro packaging.

The plasmid vector that can be used herein is not particularly limitedas long as it can be replicated and maintained in the host. Phage vectoris not also particularly limited as long as it can proliferate in thehost. Cloning vectors include, for example, pBR322, pUC19, λgt10, λgt11and the like. Moreover, upon subjecting to immunological screening, thevector has preferably a promoter that enables the expression of anSRCL-P1 gene in the host.

To incorporate cDNA into a plasmid, the method of Maniatis et al.(Molecular Cloning, A Laboratory Manual, second edition) and the likecan serve as a reference. Further, to incorporate cDNA into a phagevector, the method disclosed in Hyunh et al. (DNA cloning, a practicalapproach, 1, 49, 1985) and the like can serve as a reference.

As the method for introducing the expression vector described above intohost cells, methods for example, transfection by lipopolyamine method,DEAE-dextran method, Hanahan method, lipofectin method, calciumphosphate method; microinjection, and electroporation (MolecularCloning, A Laboratory Manual, second edition) and the like may beinvolved. In vitro packaging can be readily effected by usingcommercially available kits (manufactured by Stratagene, or Amersham).

The method for the isolation of cDNA encoding SRCL-P1 protein from acDNA library prepared as described above may involve a general method,which may be used in combination, for the screening of cDNA. Forexample, a probe labeled with ³²P is produced, and a clone containingthe desired cDNA can be screened by a colony hybridization method (Proc.Natl. Acad. Sci. USA, 72, 3961, 1975), or a plaque hybridization method(Molecular Cloning, A Laboratory Manual, second edition, Cold SpringHarbor Laboratory, 2, 108, 1989). Further, a clone may be selected by aPCR method. Additionally, the desired clone can be selected through useof an antibody that recognizes SRCL-P1 when a cDNA library is producedusing a vector that can express cDNA.

Furthermore, when an SRCL-P1 gene is isolated from cells that expressSRCL-P1 gene, for example, the expressing cells are dissolved using SDSor proteinase K, followed by a phenol treatment. Unwanted RNA isdigested with ribonuclease. Thus resultant DNA is digested withrestriction enzyme, and the resulting DNA fragments are amplified usingphage or cosmid to produce a library. Thereafter, the desired clone isselected, and then an SRCL-P1 gene can be obtained.

The nucleotide sequence of the DNA obtained accordingly can bedetermined by a Maxam-Gilbert method (Proc. Natl. Acad. Sci. USA, 74,560, 1977) or a Sanger's method (Proc. Natl. Acad. Sci. USA, 74, 5463,1977). The SRCL-P1 gene can be obtained by excising from the clone asobtained above.

Through use of the primer synthesized on the basis of the nucleotidesequence of SRCL-P1, cloning can be also effected by a RT-PCR methodusing poly(A)^(□) RNA of the cells expressing SRCL-P1 as a template.Further, the desired cDNA can be also obtained by directly screening thecDNA library after producing/synthesizing a probe based on thenucleotide sequence of SECL-P1, not by way of the PCR. The gene of thepresent invention can be selected among the genes obtained by themethods described above through the verification of the nucleotidesequence of the gene. The gene of the present invention can be alsoproduced according to the conventional method in which chemicalsynthesis of a nucleic acid, e.g., phosphoimidite method (Mattencci, M.D. et al., J. Am. Chem. Soc., 130, 3185, 1981) or the like, is employed.

Method for Producing Expression Vector

The present invention also relates to a vector comprising a nucleotidesequence of SRCL-P1s. The vector is not particularly limited so far asit can express the SRCL-P1s protein, for example, a plasmid vector, anRNA vector, a DNA vector, a virus vector, a phage vector and the likemay be employed. Specifically, examples thereof include pBAD/H is,pRSETA, pcDNA2.1, pTrcHis2A, pYES2, pBlueBac4.5, pcDNA3.1 or pSecTag2manufactured by Invitrogen, pET or pBAC manufactured by Novagen Co.,pGEM manufactured by Promega, pBluescriptII, pBS, Phagescript, pSG orpSV2CAT manufactured by Stratagene, or pGEX, pUC18/19, pBPV, pSVK3 orpSVL manufactured by Pharmacia Co.

The cDNA sequence of SRCL-P1s ligated to the expression vector isoperatively linked to a promoter. The promoter includes for example,phage λPL promoter, E. coli lac, trp, tac promoter, SV40 early and latepromoter, T7 and T3 promoter, retrovirus LTR promoter. Specifically, theprompter for use in eukaryotic cells include CMV promoter, HSV promoter,SV40 early and late promoter, retrovirus LTR promoter, RSV promoter,metallothionein promoter. In addition, the expression vector may containa marker to allow the selection of the transformed host, and anenhancer. Examples of the marker include dihydrofolate reductase gene,neomycin resistant gene, ampicillin resistant gene and the like.Examples of the enhancer include SV40 enhancer, cytomegalovirus earlyenhancer promoter, adenovirus enhancer and the like.

Method for Producing Transformed Cells

The present invention further provides transformed cells carrying anucleotide sequence of the present invention to allow the expressionthereof by means of the vector as described above carrying thenucleotide sequence. The host cell for use as a transformed cell in thepresent invention may preferably include animal cells and insect cells,however, included may be any of the cells (microorganisms may be alsoincluded), which can express SRCL-P1s protein in the expression vectorof the present invention.

Exemplary animal cells and insect cells of the present invention mayrespectively include cells derived from human, or cells derived from flyor silkworm (Bombyx mor). For example, CHO cells, COS cells, BHK cells,Vero cells, myeloma cells, HEK293 cells, HeLa cells, Jurkat cells, mouseL cells, mouse C127 cells, mouse FM3A cells, mouse fibroblast,osteoblast, chondrocyte, S2, Sf9, Sf21, High Five™ cells may beincluded. The microorganism according to the present invention includeEscherichia coli, Saccharomyces cerevisiae and the like. For theintroduction of a vector into such hosts, the method as described abovemay be employed.

In regard to SR pathway involved in the onset of arteriosclerosis andthe like, SR-expressing cells of the present invention can be used foranalyzing the specificity of modified LDLs that are incorporated intocells from this pathway. In addition, they are useful as models for theanalysis of incorporation of substances into the cells via a receptor.Moreover, the cells of the present invention can be used for screeningdrugs in the process of developing therapeutic drugs ofarteriosclerosis, for example, depressants of LDL modification,inhibitors of acyl Co-A cholesterol acyltransferase (ACAT) activity, andthe like. Furthermore, they can be used for the manufacture of human SRprotein having a carbohydrate chain. They may be also employed inexperimental systems for the process of treating foreign substances ordenaturated substances via SR, or in systems for investigating infectionof B type viruses that cause infection concomitant with a modifiedalbumin.

Method for Obtaining Protein

The present invention also relates to a method for the production ofSRCL-P1 which comprises culturing a cell transformed with the nucleotidesequence of the present invention as set forth above, and harvestingthus produced SRCL-P1. Cell culture, isolation of the protein, andpurification may be carried out with conventionally known methods.

The protein of the present invention can be expressed as a recombinantfusion protein, which can be readily isolated, purified, and recognizedper se. The recombinant fusion protein is a protein expressed by addingan appropriate peptide chain to the N-terminal end and/or C-terminal endof a protein expressed from a nucleotide sequence encoding the desiredprotein. In order to facilitate the purification of the expressedprotein, the protein may be expressed as a fusion protein having asignal for extracellular secretion. In addition, the protein can beobtained from several kinds of sources such as cultured cells, culturedtissues, transformed cells and the like using conventionally knownmethods, for example, known purification methods including: salting outsuch as ammonium sulfate precipitation technique and the like; gelfiltration technique such as Sephadex and the like; ion exchangechromatographic technique; hydrophobic chromatographic technique; dyegel chromatographic technique; electrophoresis technique; dialysis;ultrafiltration technique; affinity chromatographic technique; highperformance liquid chromatographic technique; and the like.

Method of the Utilization of Gene

Probes for detecting SRCL-P1 gene can be specified on the basis of thenucleotide sequence set out in either SEQ ID NO: 1 or 3. Alternatively,primers can be specified for the amplification of DNA or RNA includingsuch a nucleotide sequence. To specify a probe or a primer based on agiven sequence is ordinarily carried out by those skilled in this art.An oligonucleotide having a specified nucleotide sequence can beobtained through chemical synthesis. When a suitable label is added tothe oligonucleotide, it can be utilized for hybridization assay inseveral formats. Alternatively, it can be also utilized in reactions forsynthesis of nucleic acids such as PCR. The oligonucleotide that isutilized as a primer is of at least 10 bases in length, and preferablyof 15 to 50 bases in length. It is desirable that the oligonucleotideused as a probe be of from 100 bases to its full length. Further, sucholigonucleotides can be also used for the diagnosis of diseases causedby mutation of an SRCL-P1 gene because they can be used for detectinggenetic mutation encoding SRCL-P1 protein and for detecting SNP. Theyare expected to be available for the diagnosis of a variety of diseasesincluding for example, arteriosclerosis, diabetic complications andAlzheimer's disease, hyper β-lipoproteinemia, hypercholesterolemia,hypertriglyceridemia, hypo α-lipoproteinemia, transplantation,atherectomy, post angiogenic restenosis, bacterial infections, and thelike. In addition, they are also useful for gene therapy whereby SRCL-P1gene is introduced into a living body to allow the expression thereof.

Moreover, it is also possible to obtain a promoter region and anenhancer region of the SRCL-P1 gene that is present in a genome, basedon a cDNA nucleotide sequence of SRCL-P1 provided by the presentinvention. In particular, these control regions can be obtained bysimilar methods to those disclosed in Japanese unexamined patentpublication No. 6-181767; J. Immunol., 155, 2477, 1995; Proc. Natl.Acad. Sci, USA., 92, 3561, 1995, and the like. Promoter region referredto herein means a DNA region which controls the expression of a genethat exists upstream of a transcription initiation site. Enhancer regionherein means a DNA region that enhances the expression of a gene thatexists in an intron, a 5′-untranslated region, or a 3′-untranslatedregion.

Method of the Utilization of Protein

SRCL-P1s proteins of the present invention can be utilized in theelucidation of functions of macrophage and fundamental immunity, theelucidation of mechanisms of development of various types of diseasesincluding for example, arteriosclerosis, diabetic complications andAlzheimer's disease, hyper β-lipoproteinemia, hypercholesterolemia,hypertriglyceridemia, hypo α-lipoproteinemia, transplantation,atherectomy, post angiogenic restenosis and the like, as well asdiagnostic, prophylactic, therapeutic methods thereof, and in thedevelopment of reagents and drugs for the same. Furthermore, they can beused as an antigen for producing antibodies to SRCL-P1s. Additionally,they can be utilized in the screening method of agonists or antagonists.

Agonist and Antagonist

The present invention also relates to agonists which stimulate theactivity or the activation of SRCL-P1 of the present invention. Inaddition, the present invention also relates to antagonists whichinhibit the activity or the activation of SRCL-P1 of the presentinvention. For screening the antagonist, a competitive experimentalsystem can be used, for example, in which 0xLDL or an antibody, and acandidate inhibitor are subjected to the interaction with cellsexpressing SRCL-P1 protein thereby allowing the candidate inhibitor toscreen based on the binding ratio of OxLDL. Otherwise, conventionallyknown methods may also be carried out to effect the screening. Further,the antagonists also include antisense nucleic acids that inhibit theexpression of SRCL-P1 gene. Included in the examples of the othermethods for the screening may be methods in which a change inextracellular pH is measured, which is caused by the activation of areceptor (Science, 246, 181-296, 1989).

The antagonist thus screened can be also utilized as a drug for thetreatment, which may include therapy, prophylaxis and the like, ofpathological states involving in the accumulation of oxidized LDL, or inthe binding of AGE to cells. The screening method comprises the steps ofcomparing the amount of binding between SRCL-P1 of the presentinvention, and oxidized LDL or AGE in the presence and absence of acandidate drug; and identifying the candidate drug for treating theintended pathological state by an inhibitory ability of the candidatedrug to the binding therebetween.

Transgenic Non-Human Animal

The present invention relates to transgenic non-human animals having analtered expression level of SRCL-P1 gene. SRCL-P1 gene herein includescDNA, genomic DNA or synthesized DNA encoding hSRCL-P14 or SRCL-P1. Forexpression of a gene, any one of the steps of transcription andtranslation should be comprised. The transgenic non-human animalsaccording to the present invention are useful for the investigation offunctions or expression mechanisms of SRCL-P1, elucidation of mechanismsof diseases that are anticipated to be involved in SRCL-P1, developmentof diseased animal models for use in screening and safety tests ofpharmaceutical products.

In the present invention, the gene can be artificially modified toincrease or decrease the expression level in comparison with the nativeexpression level of the gene by introducing mutation such as deletion,substitution, addition and/or insertion into a part of some key sites(enhancer, promoter, intron and the like) that regulate the expressionof the gene to be proper. The introduction of the mutation can becarried out by known methods to obtain a transgenic animal.

Transgenic animals in their narrow means refer to animals having germcells into which a foreign gene was artificially introduced by a geneticrecombination technique. In their broader means, they include: antisensetransgenic animals having a particular gene of which function wassuppressed using an antisense RNA; knockout animals having a particulargene knocked out using embryonic stem cells (ES cell); and animalshaving point mutation of DNA introduced, all of which are animals havinga chromosome with a foreign gene being stably introduced at an earlystage of the development of the individual, and having a genotype thatcan be transmitted to the progeny thereof.

Transgenic animals referred to herein should be comprehended in theirbroader means including all vertebrates other than human. The transgenicanimals according to the present invention are useful for theinvestigation of functions or expression mechanisms of SRCL-P1,elucidation of mechanisms of diseases that are involved in cellsexpressed in human, development of diseased animal models for use inscreening and safety tests of pharmaceutical products.

Method for producing a transgenic mouse may include: a method in which agene is directly injected into a nucleus of an ovum in a anteriornucleus phase with a micropipette under a phase contrast microscope(microinjection technique, U.S. Pat. No. 4,873,191); a method in whichembryonic stem cells (ES cells) are used. Alternatively, a method inwhich a gene is introduced into a retrovirus vector or an adenovirusvector followed by infection into an ovum; a sperm vector technique inwhich a gene is introduced into an ovum via a sperm; and the like havebeen developed.

The sperm vector technique is a genetic recombinant method in which aforeign gene is attached to a sperm, or a foreign gene is introducedinto a sperm cell with an electroporation technique, and then theforeign gene is introduced into an ovum by fertilizing the ovum (M.Lavitranoet et al., Cell, 57, 717, 1989). Alternatively, site directedgenetic recombination in vivo may be also employed by a cre/locPrecombinase system of bacteriophage P1, a FLP recombinase system ofSaccharomyces cerevisiae, or the like. Additionally, a method has beenalso reported in which a transgene of a desired protein is introducedinto a non-human animal using retrovirus.

Method for the production of a transgenic animal with a microinjectiontechnique is carried out as described below, for example.

First, a transgene is required, which is substantially constituted froma promoter involved in expression control, a gene encoding a specifiedprotein, and a poly(A) signal. The manner of the expression and/or theexpression level of a specified molecule may be affected by the promoteractivity. In addition, because transgenic animals are different amongthe produced lineages in respect to the number of the copies of theintroduced transgene, or the introduced site in the chromosome, themanner of the expression and/or the expression level must be confirmedfor each of the lineages. Since it has been elucidated that theexpression level is altered depending on the untranslated region orsplicing, an intron sequence to be spliced at a preceding site of poly(A) signal may be previously introduced. It is important to use a gene,which is introduced into a fertilized ovum, has as high purity aspossible. The animal to be used may include mice for use in collectingfertilized ova (5-6 weeks old), male mice for use in mating, femalepseudopregnant mice, vas deferens ligated male mice, and the like.

In order to efficiently obtain the fertilized ova, gonadotropin or thelike may be used for inducing the ovulation. The fertilized ova areharvested, and thereafter, a gene in an injection pipette is introducedinto a male pronucleus of the ovum by a microinjection technique. Ananimal (a pseudopregnant mouse or the like) for use in repositioning theinjected ova to an oviduct is provided, to which 10-15 ova aretransplanted per one animal. Thereafter, the born mouse can be examinedfor the introduction of the transgene by: extracting genomic DNA fromthe end portion of the tail; and detecting the transgene by a Southernmethod or a PCR technique, alternatively by a positive cloning techniquewhere a marker gene is inserted which is activated upon only theoccurrence of homologous recombination. Moreover, in order to ascertainthe expression of the transgene, a transcription product derived fromthe transgene is detected by a Northern method or a RT-PCR technique.Alternatively, a western blotting method may be carried out with aspecific antibody to the protein or a fragment thereof.

Knockout Mouse

The knockout mouse according to the present invention is one that wastreated in a manner to deprive the function of SRCL-P1 gene. Knockoutmouse refers to a transgenic mouse in which an arbitrary gene isdestroyed by a homologous recombination technique to impair thecorresponding function. The knockout mouse can be produced by homologousrecombination using ES cells, followed by the selection of the embryonicstem cell having one of the allelic gene altered/destroyed. A chimericmouse, which carry cells derived from the embryonic stem cells and cellsderived from the embryo being mixed, may be obtained by, for example,injecting the embryonic stem cell that had been genetically engineeredat blastocyst stage or morulae stage of the fertilized ovum. When thischimeric mouse (chimera refers to a single individual built-up withsomatic cells on the basis of more than two fertilized ova) is crossbredwith a normal mouse, a heterozygotic mouse can be produced having one ofthe allelic gene is entirely altered/destroyed. Further, a homozygoticmouse can be produced by crossbreeding heterozygotic mice each other.

Homologous recombination refers to the recombination that is caused by amechanism of genetic recombination between two genes having identical orextremely similar nucleotide sequences. For the selection of cells withthe homologous recombination, PCR can be employed. PCR reaction, inwhich primers corresponding to a part of the inserted gene and a part ofthe region expected to be inserted are used, may be carried out toreveal the homologous recombination occurring in cells that could yieldthe amplification products. Also, when the homologous recombination iscaused to a gene expressed in embryonic stem cells, the gene to beintroduced may be joined to a neomycin resistant gene to allow theselection after the introduction into cells by making them resistant toneomycin. Accordingly, known methods and the modified methods thereofcan be employed to enable the easy selection.

Method for Producing Antibodies

The present invention further provides antibodies that recognize SRCL-P1or fragments thereof. The antibodies in accordance with the presentinvention include for example, the antibodies to a protein comprising anamino acid sequence set out in SEQ ID NO: 2 or 4, or a fragment thereof.The antibodies (e.g., polyclonal antibodies, monoclonal antibodies,peptide antibodies) or antisera to SRCL-P1 or a fragment thereof can beproduced using SRCL-P1 or a fragment thereof of the present invention asan antigen according to any method for producing the antibodies orantisera which is known per se. In particular, the antibodies that cancontrol the function of SRCL-P1 (e.g., antibodies that recognize CRD, acollagen like domain and an α-helical coiled coil domain or the like)are useful for pharmaceutical products containing the antibody.

SRCL-P1 or a fragment thereof according to the present invention may beadministered neat or with a diluent or a carrier to a warm-bloodedanimal at a site that enables the production of the antibody upon theadministration. In order to facilitate the production of antibodies uponthe administration, complete Freund's adjuvant or incomplete Freund'sadjuvant may be administered. The administration may be usuallyconducted once per 1 to 6 weeks, and two to ten times in total. Thewarm-blooded animal used may include for example, monkey, rabbit, dog,guinea pig, mouse, rat, sheep, goat, chicken, and the like. Among these,mouse and rat may be preferably used. Rat that may be preferably usedincludes Wistar and SD strain rat, and mouse that may be preferably usedincludes BALB/c, C57BL/6 and ICR strain mouse and the like.

Upon the production of cells that produce a monoclonal antibody, anindividual with the antibody titer that can be recognized therein isselected from the warm-blooded animals e.g., mice that had beenimmunized with an antigen. On two to five days after final immunization,spleen or lymph node is collected, and the antibody producing cellscontained therein are subjected to the fusion with myeloma cells toeffect the preparation of monoclonal antibodies producing cells. Thedetermination of the antibody titer in the antiserum may be carried outfor example, by subjecting a labeled SRCL-P1 described below to areaction with the antiserum, and measuring the activity of the labelbound to the antibody. The fusion operation can be performed inaccordance with a known technique for example, the method of Kohler andMilstein (Nature, 256, 495, 1975) and the modified method thereof (J.Immunol. Method, 39, 285, 1980; Eur. J. Biochem., 118, 437, 1981;Nature, 285, 446, 1980). Examples of the fusion accelerating agent mayinclude polyethylene glycol (PEG), Sendai virus and the like, andpolyethylene glycol may be preferably used. In addition, lectin,poly-L-lysine or DMSO may be added ad libitum to raise the efficiency ofthe fusion.

Examples of the myeloma cell include X-63Ag8, NS-1, P3U1, SP2/0, AP-1and the like, and SP2/0 may be preferably used. The ratio of antibodyproducing cell (spleen cell) number to myeloma cell number preferablyused is 1:20-20:1. PEG (preferably, PEG1000-PEG6000) is added atapproximately 10-80%. The fusion mixture is incubated at 20-40° C.,preferably at 30-37° C. for 1-10 min. Such a condition enables efficientcell fusion. Screening of the hybridoma that produces anti-SRCL-P1antibody may be performed by using various methods, which include forexample, a method in which a supernatant of hybridoma culture is addedto a solid phase (e.g., a microplate) absorbed with SRCL-P1 antigendirectly or with a carrier, and then an anti-immunoglobulin antibody(when the cell used for the cell fusion was derived from a mouse,anti-mouse immunoglobulin antibody may be used) that was labeled with aradioactive substance, enzyme or the like, or protein A is added theretothereby detecting the anti-SRCL-P1 antibody bound to the solid phase; ora method in which a supernatant of hybridoma culture is added to a solidphase absorbed with anti-immunoglobulin antibody or protein A, and thenSRCL-P1 labeled with a radioactive substance, enzyme or the like isadded thereto thereby detecting the anti-SRCL-P1 monoclonal antibodybound to the solid phase.

Selection and cloning of the anti-SRCL-P1 antibody can be carried out byknown methods per se, or the modified methods thereof. Usually, themethod is carried out in a medium for animal cells added with HAT(hypoxanthine, aminopterin, thymidine). The medium for use in theselection, cloning and growing may be any one of the media in whichhybridoma can grow. For example, RPMI medium containing 1-20%,preferably 10-20% of fetal bovine serum, GIT medium containing 1-10% offetal bovine serum, or serum free medium for hybridoma culture, and thelike. The temperature of the culture may be preferably about 37° C. Theculture period may be usually five days to three weeks, preferably oneweek to two weeks. The culture is usually conducted in the presence of5% carbon dioxide gas. The antibody titer of the supernatant of thehybridoma culture can be measured in a similar manner to the measurementof the antibody titer of anti-SRCL-P1 antibody in an antiserum asdescribed above. In other words, a radioimmunoassay (RIA) technique, anenzyme linked immunosorbent assay (ELISA) technique, a FIA (fluorescentimmunoassay) technique, a plaque measurement technique, an agglutinationreaction technique and the like may be employed as the measurementmethod, however, the ELISA technique as described below is preferred.

The screening by an ELISA technique can be carried out in accordancewith the following procedure. A protein, which was prepared by a similarmethod to that for the immunoantigen, is immobilized on the surface ofeach well of an ELISA plate. Next, BSA, MSA, OVA, KLH, gelatin orskimmed milk or the like is immobilized for the purpose of preventingnon-specific adsorption. To each well of this plate added with asupernatant solution of the hybridoma culture, followed by allowing theimmunoreaction by standing for a predetermined time. Each well is washedusing a washing solution such as PBS or the like. Surfactant may bepreferably added to this washing solution. An enzyme-labeled secondaryantibody is added, and the mixture is allowed to stand for apredetermined time. The enzyme for labeling which can be used includesβ-galactosidase, alkaline phosphatase, peroxidase and the like. Afterthe washes with the same washing solution, enzyme reaction is effectedthrough adding a substrate solution of the labeled enzyme that wasemployed. When the desired antibody is present in the added supernatantsolution of the hybridoma culture, the enzyme reaction proceeds tochange the color of the substrate solution.

Cloning can be usually carried out by known methods per se, such as asemisolid agar technique, a limiting dilution technique or the like.Specifically, after the well in which the desired antibody is producedis confirmed by the method described above, a single clone is obtainedthrough conducting the cloning. The method for cloning may involve alimiting dilution technique or the like, in which hybridoma cells arediluted so that one colony per one well of a culture plate is formed,and thereafter the culture is conducted. Cloning by a limiting dilutiontechnique may be performed through the use of feeder cells in order toelevate the colony formation ability, otherwise, a cell growth factorsuch as interleukin 6 may be added thereto. Alternatively, FACS andsingle cell manipulation techniques can be employed for the cloning. Thecloned hybridoma is cultured preferably in a serum free medium, and anappropriate amount of the antibody is added to the supernatant thereof.Thus resulting single hybridoma may be subjected to a large scaleculture using a flask or a cell culture equipment, or may be cultured inthe peritoneal cavity of an animal (J. Immunol. Meth., 53, 313, 1982) togive a monoclonal antibody. When the culture is conducted in a flask, amedium for cell culture (IMDM, DMEM, RPMI 1640, MEM and the like)containing 0-20% of FCS can be used. When the culture is conducted inthe peritoneal cavity of an animal, an animal of the same species, andthe same strain as the animal from which myeloma cells derived that wereused for the cell fusion; otherwise an athymic nude mouse may bepreferably used. Hybridoma is transplanted after mineral oil such aspristine or the like is previously administered to the animal. Ascitescontaining the monoclonal antibody can be obtained after one to twoweeks passed, when the myeloma cells enough proliferate.

The monoclonal antibody of the present invention can be obtained as theantibody, which does not cross-react with other proteins, by selectingone which recognizes an epitope specific for SRCL-P1. In general, anepitope, which is presented by serial amino acid residues of at leastmore that or equal to five, preferably 7 to 20 amino acids among theamino acid sequence constituting the protein, is referred to as anepitope inherent in the protein. Therefore, the monoclonal antibody thatrecognizes an epitope constituted from a peptide having an amino acidsequence, which were selected from the amino acid set out in any of SEQID NO: 2 and 4, consisting of at least five serial amino acid residuesmay be identified as the monoclonal antibody specific to hSRCL-P1 ormSRCL-P1 according to the present invention. When an amino acid sequenceis chosen which is conserved among the amino acid sequence set out inSEQ ID NO: 2 and 4, an epitope common to SRCL-P1 can be selected.Alternatively, a monoclonal antibody can be selected which enables thediscrimination of each protein, with a region including an amino acidsequence specific for each of the sequences.

The separation and purification of anti-SRCL-P1 monoclonal antibody canbe carried out according to the separation and purification method of animmunoglobulin similarly to the usual separation and purification methodof the polyclonal antibodies. Known purification method which can beadopted may include for example, a salt precipitation technique, analcohol precipitation technique, an isoelectric point precipitationtechnique, an electrophoretic technique, an ammonium sulfateprecipitation technique, an adsorption/desorption technique by an ionexchanger (e.g., DEAE), an ultracentrifugation technique, a gelfiltration technique, and a specific purification technique in which anantibody alone is collected by an antigen-bound solid phase or an activeadsorbent such as protein A or protein G, or the like, followed bydissociation of the binding to give the antibody. For the purpose ofpreventing the formation of aggregates, or the decrease in the antibodytiter in the purification step, for example, human serum albumin may beadded at a concentration of 0.05-2%. Otherwise, amino acids such asglycine, α-alanine and the like, in particular, basic amino acid such aslysine, arginine, histidine and the like, saccharides such as glucose,mannitol and the like, salts such as sodium chloride may be also added.In the case of IgM, which is known to be liable to agglutinate, it maybe treated with β-propionolactone and acetic anhydride.

The polyclonal antibody according to the present invention can beproduced by known methods per se, or the modified methods thereof. Forexample, to produce a polyclonal antibody, an immunoantigen (a proteinantigen) itself or a complex, which was formed with the immunoantigenand a carrier protein, is used for the immunization of a warm-bloodedanimal in a similar manner to the method for producing the monoclonalantibody described above, followed by collecting the preparationcontaining the antibody to the protein of the present invention or afragment thereof from the warm-blooded animal, and then the antibody ispurified/isolated. In respect to the complex of an immunoantigen and acarrier protein for use in the immunization of the warm-blooded animal,the kind of the carrier protein and the mixing ratio of the carrier andhapten may be optionally determined as long as the antibody can beefficiently produced to the hapten subjected to the immunization aftercrosslinking with the carrier. Thus, any kind of the carrier protein maybe crosslinked at any ratio, however, the method in which about 0.1-20,preferably about 1-5 of bovine serum albumin, bovine thyroglobulin,hemocyanin or the like, for example, is coupled with 1 of hapten byweight may be used. In addition, various condensing agents may be usedfor the coupling of the hapten and carrier, which may includeglutaraldehyde and carbodiimide, and active ester reagents containingmaleimide active ester, thiol group, dithiopyridyl group and the like.The condensation product is administered neat or with a carrier or adiluent to a warm-blooded animal at a site that enables the productionof the antibody upon the administration. In order to facilitate theproduction of antibodies upon the administration, complete Freund'sadjuvant or incomplete Freund's adjuvant may be administered. Theadministration may be usually conducted once per 2 to 6 weeks, and threeto ten times in total. Polyclonal antibodies can be collected from theblood, ascites and the like, and preferably from the blood, of thewarm-blooded animal immunized by a method as described above. Themeasurement of the antibody titer in antiserum can be carried out in asimilar manner to the measurement of the antibody titer of the antiserumas described above. The separation and purification of the polyclonalantibody can be carried out according to the separation and purificationmethod of an immunoglobulin similarly to the separation and purificationmethod of a monoclonal antibody described above.

Method of the Utilization of Antibody

Monoclonal antibodies and polyclonal antibodies to SRCL-P1 or a fragmentthereof can be utilized in diagnosis and therapy of the diseasesrelating to the cells that are expressing SRCL-P1. SRCL-P1 or a fragmentthereof can be measured using these antibodies, on the basis of theimmunological binding with SRCL-P1 or the fragment thereof according tothe present invention. Specifically, the method for measuring SRCL-P1 ora fragment thereof using such an antibody may include for example,sandwich techniques in which a sandwich complex is detected which wasproduced by subjecting SRCL-P1 or a fragment thereof to a reaction withan antibody coupled to an insoluble support and a labeled antibody; orcompetitive techniques in which SRCL-P1 or a fragment thereof in asample is measured by subjecting labeled SRCL-P1, and SRCL-P1 or afragment thereof in a sample to a competitive reaction with the antibodyfollowed by the measurement of SRCL-P1 or a fragment thereof in a samplefrom the amount of the labeled antigen that reacted with the antibody.

Upon the measurement of SRCL-P1 or a fragment thereof by the sandwichtechnique, two-step methods in which SRCL-P1 or a fragment thereof isfirst subjected to a reaction with an immobilized antibody; thereafter,unreacted materials are completely removed by washes; and then a labeledantibody is added thereto to have the immobilized antibody—SRCL-P1labeled antibody formed, alternatively, one-step methods in which animmobilized antibody, a labeled antibody and SRCL-P1 or a fragmentthereof are mixed concurrently.

Insoluble support for use in the measurement include for example,synthetic resin such as polystyrene, polyethylene, polypropylene,polyvinyl chloride, polyester, polyacrylic acid ester, nylon,polyacetal, fluorine-contained resin and the like; polysaccharides suchas cellulose, agarose and the like; glasses; metals; and the like. Theinsoluble support may be in a variety of forms, and for example,tray-like, spherical, fibrous, cylindrical, discal, vessel-like,cell-like, tubular, and the like may be adopted. The support onto whichthe antibody had been adsorbed may be stored in cold, if necessary, inthe presence of an antiseptic agent such as sodium azide and the like.

For the immobilization of the antibody, known chemical coupling methodsor physical adsorption methods may be adopted. Chemical coupling methodincludes for example, methods in which glutaraldehyde is used; maleimidemethods in whichN-succinimidyl-4-(N-maleimidemethyl)cyclohexane-1-carboxylate andN-succinimidyl-2-maleimide acetate and the like are used; carbodiimidemethods in which 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride and the like is used. Other method includesmaleimidebenzoyl-N-hydroxysuccinimide ester methods,N-succimidyl-3-(2-pyridylthio)propionic acid methods, bisdiazobenzidinemethods, dipalmityl lysine methods. Alternatively, a complex that hadbeen formed previously by subjecting the substance to be detected to areaction with two kinds of antibodies of which epitopes are differentcan be captured by the third antibody to the antibody, which had beenimmobilized in a similar manner to those described above.

The material to be used for labeling may include enzyme, fluorescentmaterials, luminescence materials, radioactive materials, metal chelatesand the like. Examples of enzyme may include peroxidase, alkalinephosphatase, β-D-galactosidase, malate dehydrogenase, staphylococcusnuclease, delta-5-steroid isomerase, α-glycerolphosphate dehydrogenase,triose phosphate isomerase, horseradish peroxidase, asparaginase,glucose oxidase, ribonuclease, urease, catalase, glucose-6-phosphatedehydrogenase, glucoamylase, acetylcholine esterase and the like.Fluorescent materials may include for example, fluoresceinisothiocyanate, phycobilin protein, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, orthophthalic aldehyde and the like.Luminescence materials may include isoluminol, lucigenin, luminol,aromatic acridinium esters, imidazole, acridinium salts and modifiedesters thereof, luciferin, luciferase, aequorin and the like.Radioactive materials may include ¹²⁵I, ¹²⁷I, ¹³¹I, ¹⁴C, ³H, ³²P, ³⁵Sand the like. These materials are not limited thereto as long as thematerial can be used in immunological determination methods. Inaddition, low molecular weight hapten such as biotin, dinitrophenyl,pyridoxal or fluorescamine may be conjugated to the antibody.Preferably, horseradish peroxidase may be used as a labeling enzyme.This enzyme can react with many kinds of substrates, which can bereadily conjugated to the antibody by a periodic acid method.

When an enzyme is used as a labeling agent, a substrate for measuringits activity, and a color-developing agent as needed may be employed.When peroxidase is used as an enzyme, H₂O₂ may be used as a substratesolution, and 2,2′-azino-di-[3-ethylbenzthiazolin sulfonate]ammonium(ABTS),5-aminosalicylic acid, orthophenylenediamine, 4-aminoantipyrine,3,3′,5,5′-tetramethylbenzidine or the like may be used as acolor-developing agent. When alkaline phosphatase is employed as anenzyme, orthophenylphosphate, paranitrophenylphosphate or the like maybe used as a substrate. Alternatively, when β-D-galactosidase is used asan enzyme, fluorescein-di-(1-D-galactopyranoside),4-methyl-umbelliferyl-D-galactopyranoside, or the like may be used as asubstrate. The present invention also involves kit products including amonoclonal antibody, a polyclonal body described above, and reagents.

Available crosslinking agents include N,N′-orthophenylenedimaleimide,4-(N-maleimidemethyl) cyclohexanoyl N-succinimide ester,6-maleimidehexanoyl N-succinimide ester, 4,4′-dithiopyridine, and otherknown crosslinking agents. The reaction of such a crosslinking agentwith the enzyme and the antibody may be conducted in accordance withknown methods depending upon the properties of the respectivecrosslinking agents. Additionally, the antibodies to be used may be anyfragments of the antibodies for example, Fab′, Fab, F(ab′)₂ depending onthe condition. Furthermore, enzymatically labeled antibodies may beprepared by using a similar method to any one of those for polyclonalantibodies and monoclonal antibodies. When the enzymatically labeledantibody that was obtained by using the aforementioned crosslinkingagent is purified by any known methods such as affinity chromatographyor the like, more sensitive immunological determination system can beachieved. The enzymatically labeled antibody, which was purified in sucha manner, is stored in a cold and dark place after adding thimerosal,glycerol or the like as a stabilizer, alternatively, after beinglyophilized.

The subject sample for the measurement may be a sample containingSRCL-P1, which may include body fluids such as plasma, serum, blood,urine, tissue fluid, cerebrospinal fluid and the like, various types ofcells, tissues, and the like.

Method for Producing Humanized Antibody

It is ethically impermissible to produce antibodies by immunizing humanwith an optional antigen. Further, when a mouse monoclonal antibody isadministered to a human body, there is a risk of the occurrence of avariety of adverse effects, because the antibody is a heterogeneousprotein to human. Therefore, an antibody with lowered antigenicity tohuman is preferred when the antibody is administered to human.

Method for the production of human monoclonal antibodies involvestransformation techniques with Epstein-Barr virus (EBV), and fusiontechniques in which thus transformed cells and parent cells are fused;methods in which a chimeric antibody or a humanized antibody is producedusing genetic engineering techniques; and the like in addition to cellfusion techniques. Chimeric antibody refers to an antibody that wasproduced by linking immunoglobulin gene fragments from heterogeneousanimals. Humanized antibody refers to an antibody having a substitutedprimary structure in part other than a complementarity determiningregion (CDR) of H chain and L chain with the corresponding primarystructure of a human antibody through introducing the alteration to amouse antibody or the like that is heterogeneous to human.

For the production of a chimeric antibody, a mouse is immunized first,and an antibody variable region (V region) that binds to an antigen isexcised from a gene of the mouse monoclonal antibody. Thereafter, the Vregion is linked to a gene of an antibody constant region (C region)derived from human myeloma to give a chimeric gene. Upon expression ofthis chimeric gene in a host cell, human-mouse monoclonal antibody canbe produced. Because chimeric antibodies are less antigenic to human,they can be utilized as a monoclonal antibody for therapeutic use to beadministered into a human body, or for use in diagnostic imaging. Knowntechniques relevant to chimeric antibodies involve Japanese patentunexamined publication No. Hei 05-304989, Japanese patent unexaminedpublication No. Hei 04-330295, WO9106649, Japanese patent unexaminedpublication No. Sho 63-036786, Japanese patent examined publication No.Hei 06-98021, and the like.

Moreover, humanized antibodies were recently developed, which areappreciated as being more useful than chimeric antibodies. Humanizedantibody refers to an antibody that is humanized as a whole moleculeexcept for CDR of an antibody molecule by grafting only a sequence of agene for an antigen-binding site (CDR: complementarity determiningregion) of an antibody molecule into a gene of a human antibody (CDRgrafting). This antibody is appreciated as being safer with lessantigenicity than the human-mouse chimeric antibody because it has lesspart derived from a mouse antibody. When SHM-D 33 strain (ATCC CRL 1668)or RF-S1 strain, both of which being human/mouse heteromyeloma, is usedas a parent cell for producing a human monoclonal antibody, high fusionefficiency can be achieved that is equivalent to mouse parent cells.Hybridoma that was obtained using these parent cells can be clonedwithout feeder cells, and it can produce IgG type antibody in acomparatively stable manner at a large amount. For the culture of theparent cells, ERDF medium supplemented with 15% FCS may be used,although other operation may be similarly carried out to the operationfor the murine cells. Additionally, in order to produce an IgG typehuman monoclonal antibody, human lymphocytes collected from peripheralblood may be preferably employed, which were sufficiently sensitizedwith an antigen. When it is difficult to obtain sufficiently sensitizedlymphocytes, sensitization with an antigen may be also conducted invitro. In Japan, clinical trials have been currently carried out forhumanized antibodies to adult T cell leukemia. In respect to theproduction of human antibodies and the related art, for example,reference should be made to those disclosed in Genentech Inc., USA(WO9222653, WO9845332, WO9404679, WO9837200, WO9404679) and CelltechInc., England (WO9429451, WO9429351, WO9413805, WO9306231, WO9201059,WO9116927, WO9116928, WO9109967, WO8901974, WO8901783), and the like.

Using the methods and the like described above, the antibodies accordingto the present invention can be humanized, and such antibodies would beextremely useful upon the administration to human.

Composition

The SRCL-P1 polynucleotides or proteins and antibody substances, andantagonists and the like of SRCL-P1 are possibly utilized in diagnostic,prophylactic and therapeutic methods, and for the development ofreagents and drugs for various types of diseases involved in theaccumulation of oxidized LDL (modified LDL) including for example,arteriosclerosis and the like, as well as disorders involved in thebinding of AGE to cells such as glomerulosclerosis and the like,diabetic complications and AD, hyper β-lipoproteinemia,hypercholesterolemia, hypertriglyceridemia, hypo α-lipoproteinemia,transplantation, atherectomy, and post angiogenic restenosis, bacterialinfections and the like. Further, the ingredient can be combined orblended with known medical drugs. For example, the ingredient can becombined or blended with therapeutic drugs of atherosclerosis, e.g.,ACAT inhibitors, HMG-CoA reductase inhibitors, lipid regulants, bileacid regulants.

Pharmaceutical composition according to the present invention maycomprise SRCL-P1 polynucleotides or proteins, substances that stimulateor inhibit the activity or activation of SRCL-P1 protein, substancesincluding antibodies to SRCL-P1 protein and the like (hereinafter,referred to as “SRCL-P1 related substance”). The SRCL-P1 relatedsubstances can be used neat, or after subjecting to several kinds oftreatment such as dilution in water and the like, and they may also beused after blending in pharmaceutical products, quasi drugs and thelike. In these cases, the amount of the substance to be blended may bedetermined ad libitum. When the substance is formulated for the systemicadministration, 0.001-50% by weight, in particular, 0.01-10% by weightis permissible. When the amount is less than 0.001%, sufficient actionof lacrimation may not be enabled. When the amount is greater than 50%,properties such as stability, flavor and the like of the compositionitself may be deteriorated.

The route of administration can be optionally selected from theadministration via mucosa, transdermal administration, intramuscularadministration, subcutaneous administration, endorectal administration,topical ocular administration, and the like, in addition to oraladministration and intravenous administration described above.

The SRCL-P1 realated substance according to the present invention may beincluded in the formulation as a salt. Pharmaceutically acceptable saltsinclude for example, salts with base such as inorganic base, organicbase and the like; acid addition salts such as those of inorganic acid,organic acid, basic or acidic amino acid. Inorganic bases include forexample, alkaline metal such as sodium, potassium and the like; alkalineearth metal such as calcium, magnesium and the like; aluminum, ammoniumand the like. Organic bases include for example, primary amines such asethanolamine and the like; secondary amines such as diethylamine,diethanolamine, dicyclohexylamine, N,N′-dibenzylethylenediamine and thelike; tertiary amines such as trimethylamine, triethylamine, pyridine,picoline, triethanolamine and the like. Inorganic acids include forexample, hydrochloric acid, hydrobromic acid, nitric acid, sulfuricacid, phosphoric acid and the like. Organic acids include for example,formic acid, acetic acid, lactic acid, trifluoroacetic acid, fumaricacid, oxalic acid, tartaric acid, maleic acid, benzoic acid, citricacid, succinic acid, malic acid, methanesulfonic acid, ethanesulfonicacid, benzenesulfonic acid, p-toluenesulfonic acid and the like. Basicamino acids include for example, arginine, lysine, ornithine and thelike. Acidic amino acids include for example, aspartic acid, glutamicacid and the like.

Examples of dosage forms for use in oral administration include powderedformulations, granulated formulations, encapsulated formulations, pills,tablets, elixirs, suspensions, emulsions, syrups and the like, which maybe selected ad libitum. In addition, such formulations may be modified,which may involve release control, stabilization, facilitation ofdisintegration, blocking of disintegration, enteric coating,facilitation of absorption and the like. Moreover, examples of dosageforms for the intraoral topical administration include chewableformulations, sublingual formulations, buccal formulations, lozenges,ointments, plasters, liquid formulations and the like, which may beselected ad libitum. Further, such formulations may be modified, whichmay involve release control, stabilization, facilitation ofdisintegration, blocking of disintegration, enteric coating,facilitation of absorption and the like.

Known drug delivery system (DDS) techniques may be applied to dosageforms as described above. DDS formulation referred to herein involvessustained release formulations, topically applied formulations(lozenges, buccal formulations, sublingual formulations), drugcontrolled release formulations, enteric coated formulations,formulations soluble in stomach and the like, which are formulationsthat are prepared so that most appropriate dosage form is accomplishedtaking into account of the administration route, bioavailability,adverse effect and the like.

Components for DDS essentially comprise a drug, a drug release module, acoating and a therapy program. In detail, the drug having a short halflife is preferred, which permits rapid decline of the bloodconcentration particularly upon cessation of the release thereof. Thecoating is preferably nonreactive to the body tissue of the part towhich the drug is administered. In addition, the therapy program ispreferably configured so that the most optimal drug concentration iskept during the predetermined period. The drug release modulesubstantially has a drug storage, a release control part, an energysource, and a release opening or a release surface. All of thesefundamental components are not necessarily required, and thus addition,deletion or the like may be optionally carried out to select the bestmode.

Examples of materials which can be used for DDS include polymers,cyclodextrin derivatives, lecithin and the like. The polymer may includeinsoluble polymers (silicone, ethylene-vinyl acetate copolymer,ethylene-vinyl alcohol copolymer, ethylcellulose, cellulose acetate andthe like), water soluble polymers and hydroxyl gel-forming polymers(polyacrylamide, polyhydroxyethyl methacrylate cross-linked form,polyacryl cross-linked form, polyvinyl alcohol, polyethyleneoxide, watersoluble cellulose derivatives, cross-linked poloxamer, chitin, chitosanand the like), slow dissolving polymers (ethyl cellulose, a partialester of methylvinyl ether-maleic anhydride copolymer and the like),polymers soluble in stomach (hydroxylpropylmethyl cellulose,hydroxylpropyl cellulose, carmellose sodium, macrogol,polyvinylpyrrolidone, dimethylaminoethyl methacrylate-methylmethacrylate copolymer and the like), enteric polymers(hydroxylpropylmethyl cellulose phthalate, cellulose acetate phthalate,hydroxylpropylmethyl cellulose acetate succinate, carboxymethylethylcellulose, acrylic acid polymers and the like), biodegradable polymers(heat coagulation or cross-linked albumin, cross-linked gelatin,collagen, fibrin, polycyanoacrylate, polyglycolic acid, polylactic acid,poly β-hydroxyacetic acid, polycaprolactone and the like), which can beselected ad libitum on the basis of the dosage form.

In particular, silicone, ethylene-vinyl acetate copolymer,ethylene-vinyl alcohol copolymer, a partial ester of methylvinylether-maleic anhydride copolymer can be used for the control of drugrelease; cellulose acetate can be used as a material of a osmoticpressure pump; ethyl cellulose, hydroxypropylmethyl cellulose,hydroxypropyl cellulose, methyl cellulose can be used as a material of amembrane of slow dissolving formulations; and polyacryl cross-linkedform can be used as an attaching agent to oral mucosa or ophthalmicmucosa.

Further, the formulation can be manufactured with adding solvent,excipient, coating agent, base, binding agent, lubricant, disintegrant,solubilizing agent, suspending agent, thickening agent, emulsifyingagent, stabilizing agent, buffering agent, isotonizing agent, soothingagent, preservative agent, flavoring agent, fragrance agent, coloringagent and the like in compliance with its dosage form (known dosagesform such as forms for oral administration, injection, suppository andthe like).

Although specific examples are respectively illustrated below, theseexamples should not be construed as limiting the present invention.

[solvent] purified water, water for injection, saline, peanut oil,ethanol, glycerol;

[excipient] starches, lactose, glucose, sucrose, crystalline cellulose,calcium sulfate, calcium carbonate, talc, titanium oxide, trehalose,xylitol;

[coating agent] sucrose, gelatin, cellulose acetate phthalate andpolymers as described above;

[base] vaseline, vegetable oil, macrogol, base for oil in wateremulsion, base for water in oil emulsion;

[binding agent] natural polymer compounds such as starch and derivativesthereof, cellulose and derivatives thereof, gelatin, sodium alginate,gum tragacanth, gum arabic, and the like; synthetic polymers such aspolyvinylpyrrolidone and the like; dextrin, hydroxylpropyl starch;

[lubricant] stearic acid and salts thereof, talc, waxes, wheat starch,macrogol, hydrogenated vegetable oil, sucrose fatty acid ester,polyethylene glycol;

[disintegrant] starch and derivatives thereof, agar, gelatin powder,sodium bicarbonate, cellulose and derivatives thereof, carmellosecalcium, hydroxypropyl starch, carboxymethyl cellulose, and salts andderivatives thereof, poorly substituted hydroxypropyl cellulose;

[solubilizing agent] cyclodextrin, ethanol, propylene glycol,polyethylene glycol;

[suspending agent] gum arabic, gum tragacanth, sodium alginate, aluminummonostearate, citric acid, various surfactants;

[thickening agent] carmellose sodium, polyvinylpyrrolidone, methylcellulose, hydroxypropylmethyl cellulose, polyvinyl alcohol, gumtragacanth, gum arabic, sodium alginate;

[emulsifying agent] gum arabic, cholesterol, gum tragacanth, methylcellulose, various surfactants, lecithin;

[stabilizing agent] sodium bisulfite, ascorbic acid, tocopherol,chelating agent, inert gas, reducing agent;

[buffering agent] sodium hydrogenphosphate, sodium acetate, boric acid;

[isotonizing agent] sodium chloride, glucose;

[soothing agent] procaine hydrochloride, lidocaine, benzyl alcohol;

[preservative agent] benzoic acid and salts thereof, p-hydroxybenzoicesters, chlorobutanol, inverted soap, benzyl alcohol, phenol,thimerosal;

[flavoring agent] sucrose, saccharin, glycyrrhiza extract, sorbitol,xylitol, glycerol;

[fragrance agent] orange peel tincture, rose oil;

[coloring agent] water soluble edible dye, lake dye.

EXAMPLES

Novel scavenger receptor according to the present invention is describedin more detail by the following non-limiting illustrative examples.However, the present invention should not be construed to be limited bythe examples.

Specifically, search on EST database (Example 1); preparation of probesfor the screening (Example 2); screening of a cDNA library from humanplacenta (Example 3); base sequencing of novel human scavenger receptor(Example 4); and obtaining novel mouse scavenger receptor cDNA (Example5); as well as method for producing a transfectant that transientlyexpresses the novel human scavenger receptor (Examples 6 and 7); methodfor producing a transfectant that stably expresses the novel humanscavenger receptor (Examples 8 and 9); verification of the bindingspecificity of the novel human scavenger receptor (Example 10);demonstration of phagocytic capacity (Example 11); and demonstration ofexpression in vascular endothelial cells (Example 12) are describedbelow, all of which were illustrated.

Example 1 Search on EST Database

By comparing amino acid sequences of known collecting, i.e., human MBP,human SP-A and human SP-D (see, FIGS. 2 and 3, wherein, circumscribedamino acid residues denote the part that are recognized to behomologous), a region highly conserved between the molecules wassearched. Consequently, it was revealed that 27 amino acidscorresponding to from position 220 to position 246 of the human MBPamino acid sequence (FIG. 3, outlined characters, SEQ ID NO: 5) werehighly conserved. Therefore, several consensus sequences in compliancewith this region were produced, and search on EST (Expressed SequenceTags) database was conducted. EST database that was employed contained676750 sequences dated Oct. 11, 1996.

As a result, several data were obtained for highly homologous amino acidsequence to the 27 amino acids described above. The amino acid sequencesof thus resultant data were searched on GenBank/EST database, anddetermined whether they were any of known or unknown substance.Consequently, two data (accession number: W72977 and R74387) thatexhibit high homology but contain unknown nucleotide sequence could beobtained among data that were obtained when the amino acid sequence setout below was used as a consensus sequence: (SEQ ID NO: 6)Glu-Lys-Cys-Val-Glu-Met-Tyr-Thr-Asp-Gly-Lys-Trp-Asn-Asp-Arg-Asn-Cys-Leu-Gln-Ser-Arg-Leu-Ala-Ile- Cys-Glu-Phe.These data were respectively derived from placenta and fetal heart,which represent a part of the nucleotide sequence of a novel collectin.

Accordingly, a clone derived from fetal heart (I.M.A.G.E. ConsortiumClone ID 34472) was purchased from ATCC (American Type CultureCollection) among these, and utilized in the preparation of probes forthe screening to obtain a novel scavenger receptor below.

Example 2 Preparation of Probes for Screening

The nucleotide sequence of an insert DNA of the above-described clonewas sequenced with a primer (Pharmacia Co., M13 Universal Primer (SEQ IDNO: 7,5′-fluorescein-cgacgttgtaaaacgacggccagt-3′) and M13 Reverse Primer(SEQ ID NO: 8, 5′-fluorescein-caggaaacagctatgac-3′)).

Thus resulting nucleotide sequences was aligned to the amino acidsequence of an open reading flame of a collectin, and then a nucleotidesequence corresponding to the amino acid sequence that can be readtherefrom was extracted. Primers for digoxigenin (DIG) labeled cDNAprobe that correspond to a part of the extracted sequence (Reverseprimer: caatctgatgagaaggtgatg (SEQ ID NO: 9) and Forward primer:acgaggggctggatgggacat (SEQ ID NO: 10)) were produced using AppliedBiosystems Inc., 392A DNA/RNA synthesizer. DIG labeling was conductedusing a PCR DIG probe synthesis kit (Boeringer Mannheim Co., Ltd). Theconstitution of a reaction is as follows: 2 μl (100 ng) of plasmid DNA(clone W72977, 50 ng/μl); 5 μl of 10× buffer; 5 μl of 25 mM MgCl_(□); 5μl dNTP (PCR labeling mix); 2.5 μl of 20 μM Reverse primer; 5 μl of 20μM Forward primer; 28 μl of H_(□)O; 0.5 μl of Taq polymerase. PCRreaction was performed using Atto Co., Ltd., Zymoreactor, with 35 cyclesof 92° C. for 1 min, 55° C. for 1 min, and 72° C. for 2 min.

Example 3 Screening of cDNA Library Derived from Human Placenta

A phage cDNA library derived from human placenta was first subjected tothe titration as follows. A solution of 0.2 ml of Escherichia coliY1090r, which had been cultured in mLB medium (LB medium (1g triptone,0.5 g yeast extract, 0.5 g NaCl/100 ml) containing 10 mM MgSO_(□) and0.2% maltose) at 37° C. for 16 hours, and 0.1 ml of cDNA libraryserially diluted with SM buffer (5.8 g NaCl, 2g MgSO_(□).7H_(□)O, 25 mlof 2 M Tris-HCl (pH 7.5), 5 ml of 2% gelatin/L) were incubated at 37° C.for 15 min. Thereafter, the mixture was added to 2.5 ml of LB-TOPagarose (0.75% agarose/LB medium) to give a homogenous mixture, andplated on 90 mmΦ LB medium plate (Iwaki glass Co., Ltd.) (1.5% agar/LBmedium). The medium was hardened in 15 minutes at room temperature,followed by incubation at 42° C. for 5 hours. After counting the plaquenumber of each plate, the titer of phage of each plate was calculated.As a result, the titer was calculated to be 2.1×10¹⁰ pfu/ml. Thustitrated cDNA library was screened as follows using the probes producedin accordance with Example 2.

A solution of 0.6 ml of Escherichia coli Y1090r, which had been culturedin mLB medium at 37° C. for 16 hours, and 1×10^(□) pfu of the cDNAlibrary diluted with SM buffer were incubated at 37° C. for 15 min.Thereafter, the mixture was added to 7.5 ml of LB-TOP agarose (0.75%agarose) to give a homogenous mixture. Ten plates of 140 mm of LB mediumrectangular plate (Nissui Seiyaku Co., Ltd.) to which the mixture wasplated were provided, and hardened in 15 minutes at room temperature,followed by incubation at 42° C. for 5 hours. After confirming theplaque formation, transfer to a nylon membrane was conducted. Thetransfer was carried out using Nytran 13N (Schleicher and Schuell Co.).A filter of 12.5 cm×9.0 cm was immersed in distilled water, andmoisturized for 10 minutes. Thereafter, the excess moisture waseliminated on a Whatman 3MM paper, and the filter was placed on theplate with the plaques formed. After leaving to stand for two minutes,the filter was stripped off, and air dried for 10 minutes. Phage DNA wasmodified with 0.2 M NaOH/1.5 M NaCl for 2 min, and neutralized with 0.4M Tris-HCl(pH7.6)/2×SSC for 2 min, followed by washes with 2×SSC for 2min. Thereafter, the phage DNA was fixed on the membrane by ultravioletirradiation using GS GENE LINKER (Bio-Rad Inc.,). Detection of thehybridization and signals was executed as described below. The filterwas moisturized with 2×SSC, and the excess moisture was eliminated witha Whatman 3MM paper. Then the filter was placed into a hybridizationbag, and subjected to prehybridization in a hybridization solution(5×SSC, 1% blocking agent, 0.1% N-lauroylsarcosine, 0.02% SDS) at 68° C.for 1 hour. Subsequently, the hybridization solution was removed fromthe bag, and thereto added a hybridization solution that was prepared togive 10 ng/ml of DIG labeled cDNA probe. Hybridization was carried outat 55° C. for 16 hours. After the hybridization was completed, thefilter washed with 2×SSC/0.1% SDS solution for 5 min twice at roomtemperature, and with 0.5×SSC/0.1% SDS solution for 15 min twice at 55°C. Next, SDS was removed with DIG buffer I (100 mM Tris-HCl, 150 mM NaCl(pH7.5)) for 1 minute, followed by blocking of the filter with DIGbuffer II (1% blocking agent, DIG buffer I) for 30 min. After the washwith DIG buffer I for 1 min, an antibody reaction was then allowed for30 min through the addition of a solution of 5,000× anti-DIG alkalinephosphatase labeled antibody (Boeringer Mannheim Co., Ltd.) diluted withDIG buffer II. The filter washed twice with DIG buffer I for 15 min atroom temperature. The concentration of Mg²⁺ was elevated by thetreatment with DIG buffer III (100 mM Tris-HCl, 100 mM NaCl (pH 9.5), 50mM MgCl₂) for 3 min, and then a solution of NBT/BCIP (Wako Pure ChemicalIndustries, Ltd.) in DIG buffer III was added to the mixture for thecolor development. Accordingly, ten positive clones were obtained.Plaques corresponding to these clones were excised, and they wererespectively placed in a tube containing 1 ml of SM buffer. Afterstirring for 10 min, the solution was serially diluted with SM buffer.Then, 0.1 ml of thus diluted solution was mixed with a solution of 0.2ml of Escherichia coli Y1090r that had been cultured in mLB medium at37° C. for 16 hours, and the mixture was incubated at 37° C. for 15 min.Thereafter, the mixture was added to 2.5 ml of LB-TOP agarose to give ahomogenous mixture, and plated on 90 mmΦ LB medium plate. Ten platesprepared in this manner were hardened in 15 minutes at room temperature,followed by incubation at 42° C. for 5 hours. Several plaques wereobtained, which were subjected to the secondary screening similarly tothe primary screening.

Example 4 Sequencing of Nucleotide Sequence of Human Novel ScavengerReceptor

A plaque of a clone, which was deemed to be proper among the positiveclones obtained in the secondary screening, was excised from the plate.Thus resulting plaque was placed in a tube containing 200 μl ofdistilled water. After stirring for 30 min, the solution was centrifugedat 15,000 rpm for 5 min to give a supernatant.

Thus resultant supernatant was used as a template to amplify an insertDNA by PCR using TaKaRa LA PCR Kit Ver.2 (Takara Shuzo Co., Ltd.). Theconstitution of the reaction is as follows: 27 μl of the supernatant, 5μl of 10×LA PCR buffer II (Mg²⁺ free), 5 μl of 25 mM MgCl_(□), 8 μl ofdNTP mix, 2.5 μl of 20 μM λgt11 Reverse Primer (SEQ ID NO: 11,5′-ttgacaccagaccaactggtaatg-3′), 2.5 μl of 20 μM λgt11 Forward Primer(SEQ ID NO: 12, 5′-ggtggcgacgactcctggagcccg-3′), 0.5 μl of LA Taqpolymerase, H_(□)O added to adjust the total volume of 50 μl). The PCRreaction was carried out using Gene Amp PCR system 9600 manufactured byApplied Biosystems Inc., with 30 cycles of at 98° C. for 20 seconds andat 68° C. for 5 minutes. The PCR product was confirmed on 1% agarose gelelectrophoresis, and was purified by excising from the gel. Sephaglas Band Prep Kit manufactured by Pharmacia Co., was used for thepurification.

The DNA fragment excised was incorporated into pCR2.1 vector included inTA cloning kit manufactured by Invitrogen Co. The recombinant vector wastransformed into TOP10F′ cells included in the TA cloning kitmanufactured by Invitrogen Co. The transformant was cultured in LBmedium (100 μg/ml ampicillin), and then three kinds of plasmid DNA pereach clone were extracted by an alkali SDS method.

Thus resulting DNA was cut with a restriction enzyme that was envisagedto be suitable. Each DNA fragment was incorporated into pUC18 vector andtransformed into XL-1 Blue cells. The transformant was cultured in LBmedium (100 μg/ml ampicillin), and then a plasmid was extracted by analkali SDS method. Accordingly, the plasmids below were obtained: aplasmid containing an EcoR I-Hind III fragment or a Hind III-EcoR Ifragment from CL-P1-2-1; a plasmid containing an EcoR I-BamH I fragment,a BamH I-Sma I fragment, a Sma I-Hind III fragment, a Kpn I-Sau3A Ifragment, a Sau3A I-EcoR I fragment, an EcoR I-Kpn I fragment or an EcoRI-Sma I fragment from CL-P1-3-4; a plasmid containing an EcoR I-BamH Ifragment, a BamH I-Sma I fragment, a Sma I-Hind III fragment, a KpnI-Sau3A I fragment, a Sau3A I-EcoR I fragment, an EcoR I-Knp I fragmentor a Kpn I-EcoR I fragment from CL-P1-3-7. Primers for the use were: M13Universal Primer (SEQ ID NO: 7) and M13 Reverse Primer(SEQ ID NO: 8)attached to AutoRead Sequencing Kit (Pharmacia Co.,), and the followingprimers produced by using a DNA/RNA synthesizer, which were labeled withFITC (Pharmacia Co., FluorePrime), followed by sequencing of thenucleotide sequence of their entire regions with AutoRead Sequencing Kitand A.L.F. Auto Sequencer manufactured by Pharmacia Co. (SEQ ID NO: 13)HPP 1: 5′-fluorescein-cgtgaaaatgaatggaagtgg-3′, (SEQ ID NO: 14) HPP 2:5′-fluorescein-ttttatccattgctgttcctc-3′, (SEQ ID NO: 15) HPP 3:5′-fluorescein-ctggcagtccccgaggtccag-3′, (SEQ ID NO: 16) HPP 5:5′-fluorescein-gctggtccccccggagagcgt-3′.

Outline of the sequencing of the nucleotide sequences conducted as aboveis shown in FIG. 4. FIG. 4 (a) represents ORF of a collectin-likestructural part of the obtained scavenger receptor, in which G-X-Y(wherein G denotes glycine, X and Y may be any one of amino acidresidues) represents a collagen-like domain. In addition, FIG. 4 (b)represents each name of the primers described above, the nucleotidesequence that was read by a sequencer, which is shown by arrows, and M13Universal Primer (shown as “U”) and M13 Reverse Primer(shown as “R”).

Moreover, the nucleotide sequence of 5′ end region including atranscription initiation site of this sequence was determined using Capsite cDNA.

First PCR was carried out with Cap Site cDNA, Human Liver (NIPPON GENEKK) using 1RC2 Primer (5′-caaggtacgccacagcgtatg-3′ (SEQ ID NO: 17))attached thereto, and TGP1 Primer (5′-tcttcagtttccctaatccc-3′ (SEQ IDNO: 18)) that was synthesized by 392A DNA/RNA synthesizer manufacturedby Applied Biosystems Inc. The reaction mixture contained LA PCR BufferII (Mg²⁺ free), 2.5 mM MgCl₂, 1 μl of each 200 μM dATP, dCTP, dGTP anddTTP (all of which were manufactured by Takara Shuzo Co., Ltd.), CapSite cDNA Human Liver, 0.5 μM 1RC2 Primer (manufactured by NIPPON GENEKK), and 0.5 μM TGP1 Primer in a total liquid volume of 50 μl. PCR wascarried out with a program involving 35 cycles of: heat denaturation at95° C. for 20 seconds, annealing at 60° C. for 20 seconds, elongation at72° C. for 20 seconds, as well as heat denaturation at 95° C. for 5minutes prior to repeating the reaction and finally elongation at 72° C.for 10 minutes. After completing the first PCR reaction, nested PCR wascarried out. One μl of the product of the first PCR was used as atemplate, whilst primers employed were 2RC2 Primer(5′-gtacgccacagcgtatgatgc-3′ (SEQ ID NO: 19)) as attached, and syntheticTGP2 Primer (5′-cattcttgacaaacttcatag-3′ (SEQ ID NO: 20)), which wassynthesized similarly to TGP1 Primer. The reaction was conducted with asimilar constitution of the reaction and program to those of the firstPCR, except that cycle number was 25 cycles. Such a PCR reaction wascarried out with TaKaRa PCR Thermal Cycler 480 manufactured by TakaraShuzo Co., Ltd. Thus resulting PCR product was confirmed on agarose gelelectrophoresis. Thereafter, the band was excised from the gel, followedby freezing at −80° C. for 10 minutes and centrifugation at 15,000 rpmfor 10 minutes. After the centrifugation, the supernatant wasprecipitated with ethanol. Accordingly, the purification wasaccomplished.

Purified DNA fragment was incorporated into pT7Blue Vector manufacturedby Novagen Co., and thus resulting vector was transformed into competentXL1-Blue cells. The transformant was cultured in LB medium (100 μg/mlampicillin), and then a plasmid was extracted by an alkali SDS method.Nucleotide sequence was determined with AutoRead Sequencing Kit and A.L. F. DNA Sequencer manufactured by Pharmacia Co. Primers employed wereM13 Universal Primer (SEQ ID NO: 7) and M13 Reverse Primer (SEQ ID NO:8) attached to AutoRead Sequencing Kit.

Additionally, screening of a cDNA library derived from placenta(Clontech Co.,) was carried out after synthesizing a primer:5′-atcttgctgcagattcgtgac-3′ (SEQ ID NO: 21), corresponding to theupstream direction from the sequence of an N terminal portion of thecDNA clone obtained for the confirmation of the N terminus. Thescreening was carried out by PCR using a primer synthesizedcorresponding to the upstream direction: 5′-atcttgctgcagattcgtgac-3′(SEQ ID NO: 21) and a primer λgt11 5′ Sequencing Primer:5′-gactcctggagcccg-3′ (SEQ ID NO: 22) that is a part included in thevector. The reaction mixture was prepared by adding 2.5 mM MgCl₂, 1×LAPCR Buffer II (Mg²⁺ free), 2U TaKaRa LA Taq, two kinds of primers (each0.2 μM 5′-atcttgctgcagattcgtgac-3′ (SEQ ID NO: 21), λgt11 5′ SequencingPrimer: 5′-gactcctggagcccg-3′ (SEQ ID NO: 22)), 1 μl of a cDNA libraryderived from placenta to water to give the total volume of 50 μl. Thereaction was executed with one cycle of at 94° C. for 2 minutes, and 50cycles of at 94° C. for 30 seconds, at 60° C. for 30 seconds, and at 72°C. for 1 minute and 30 seconds.

Thus resulting cDNA was separated on agarose gel electrophoresis, andstained with a solution of ethidium bromide (0.1 μg/ml). Uponconfirmation of the migration pattern with a trans illuminator, theamplification of an insert corresponding to about 600 bp wasdemonstrated. Then, the amplified part was excised from the agarose gel,followed by freezing at −80° C. for 10 minutes and centrifugation at15,000 rpm for 10 minutes. After the centrifugation, the supernatant wasrecovered and subjected to the precipitation with ethanol. Accordingly,the purification was accomplished. Purified DNA fragment wasincorporated into pT7Blue Vector manufactured by Novagen Co., and thusresulting vector was transformed into competent XL1-Blue cells. Thetransformant was cultured in LB medium (50 μg/ml ampicillin), and then aplasmid was extracted by an alkali SDS method. Nucleotide sequence wasdetermined with DNA Sequencing Kit and Sequencer ABI PRISM 377manufactured by PE Applied Biosystems Inc. Primers employed were M13Universal Primer (SEQ ID NO: 7) and M13 Reverse Primer (SEQ ID NO: 8)attached to AutoRead Sequencing kit manufactured by Pharmacia Co.

Consequently, it was revealed that the sequence contained further 604bases long from the nucleotide sequence that had been obtained to theN-terminal direction. Accordingly, it was confirmed that the resultantcDNA of hSRCL-P1 obtained hereby includes 2628 bases, having ORF (openreading frame) of 2226 bases (SEQ ID NO: 1), which encodes amino acidsequence of 742 amino acids set out in SEQ ID NO: 2.

Next, search on the homology for DNA and amino acid on GenBank databasewas conducted. As a result, the resulting amino acid sequence wasrevealed to be that of a novel protein, which is distinct from anycollectin/scavenger receptors that have been found so far.

Furthermore, a mutant was obtained, having deletion of amino acidsposition 483 to 606 of the amino acid sequence set out in SEQ ID NO: 2,encoded by the nucleotide sequences position 74 to 1993 set out in SEQID NO: 23.

Example 5 Obtaining cDNA of Mouse Novel Scavenger Receptor

In a similar manner to that for hSRCL-P1, mSRCL-P1 gene could beobtained through the screening of a mouse liver cDNA library. Theresulting cDNA clone of mSRCL-P1 includes 2637 bases, having ORF (openreading frame) of 2226 bases (SEQ ID NO: 3), which was confirmed toencode amino acid sequence of 742 amino acids set out in SEQ ID NO: 4.

Example 6 Construction of Transient Expression Vector of hSRCL-P1,pEGFP-N1-hSRCL-P1

Amplification of hSRCL-P1, from initiation codon to termination codon,set out in SEQ ID NO: 1 was carried out first using a primer consistingof the nucleotide sequence: ccgctcgagcggtcaccatgaaagacgact (SEQ ID NO:25) and a primer consisting of the nucleotide sequence:tccccgcggtaatgcagatgacagtactgt (SEQ ID NO: 26) with a cDNA libraryderived from human placenta as a template by PCR (manufactured by TakaraKK: Takara Thermal Cycler MP). Thus resulting hSRCL-P1cDNA was ligatedto pT7Blue T-Vector (Novagen Co.), and transformed into Escherichiacoli, XL1-Blue. A plasmid containing hSRCL-P1cDNA was purified from theresulting clone. Following the confirmation of the nucleotide sequenceof the resulting plasmid with a sequencer, the plasmid with no error wasdigested with restriction enzymes Xho I and Sac II, and ligated topEGFP-N1 vector (Clontech Co.) that had been digested with the sameenzymes and purified. After the ligated plasmid was transformed intoEscherichia coli, XL1-Blue, the resulting clone was cultured. Theplasmid was then purified to give a transient expression vectorpEGFP-N1-hSRCL-P1.

Example 7 Expression of hSRCL-P1 Using a Transient Expression System

Transient expression in CHO cells was attempted using the expressionvector pEGFP-N1-hSRCL-P1 obtained in Example 6, and LIPOFECTAMINE 2000(LF2000) Reagent (GIBCO BRL Co.). A solution of 0.2 ml of LF2000 Reagent(LF2000 Reagent 12 μl, Nutrient Mixture F-12 Ham (Ham's F-12 medium,(manufactured by Sigma Co.))) was first prepared, and incubated at roomtemperature for 5 minutes. Then, 0.2 ml of a vector solution(pEGFP-N1-hSRCL-P1 vector 4 μg. Ham's F-12 medium) was admixedtherewith, followed by the incubation for 20 minutes. Thereafter, thesolution was added to CHO cells that had been cultured to a high densityin a 35 mm dish containing 2 ml of Ham's F-12 medium (containing 5%FCS). After incubating at 37° C. for 4 hours in the presence of 5% CO₂,the medium was replaced with a flesh medium, followed by subsequentincubation at 37° C. for 20 hours in the presence of 5% CO₂. Thepresence or absence of the expression could be confirmed by theobservation of the fluorescent image for GFP by a fluorescenceobservation system of an inverted system microscope IX70 manufactured byOlympus Co., Ltd. Thus resultant cells were identified as cellstransiently expressing hSRCL-P1.

Example 8 Construction of a Vector pcDNA3.1/Myc-H is A-hSRCL-P1 forProducing Cell Strain Stably Expressing hSRCL-P1

Amplification of hSRCL-P1, from initiation codon to termination codon,set out in SEQ ID NO: 1 was carried out first using a primer consistingof the nucleotide sequence: aatgcggccgcaccatgaaagacgacttcgcagag (SEQ IDNO: 27) and a primer consisting of the nucleotide sequence:gctctagaccgcggtaatgcagatgacagtac (SEQ ID NO: 28) with a cDNA libraryderived from human placenta as a template by PCR (manufactured by TakaraKK: Takara Thermal Cycler MP). Thus resulting hSRCL-P1cDNA was ligatedto pT7Blue T-Vector (Novagen Co.), and transformed into Escherichiacoli, XLI-Blue. A plasmid containing hSRCL-P1cDNA was purified from theresulting clone. Following the confirmation of the nucleotide sequenceof the resulting plasmid with a sequencer, the plasmid with no error wasdigested with restriction enzymes Not I and Sac II, and ligated topcDNA3.1/Myc-H is A vector (Invitorogen Co.) that had been digested withthe same enzymes and purified. After the ligated plasmid was transformedinto Escherichia coli, XLI-Blue, the resulting clone was cultured. Theplasmid was then purified to give vector pcDNA3.1/Myc-H is A-hSRCL-P1for producing a stable cell strain.

Example 9 Preparation of Cell Strain Stably Expressing hSRCL-P1

Stable expression of hSRCL-P1 was attempted using the expression vectorpcDNA3.1/Myc-H is A-hSRCL-P1 obtained in Example 8, and LIPOFECTAMINE2000 (LF2000) Reagent (GIBCO BRL Co.). A 0.5 ml solution of LF2000Reagent (LF2000 Reagent 30 μl, Ham's F-12 medium) was first prepared,and incubated at room temperature for 5 minutes. Then, 0.5 ml of avector solution (vector 10 μg, Nutrient Mixture F-12 Ham (Ham's F-12medium) (Sigma Co.)) was admixed therewith, followed by the incubationfor 20 minutes. Thereafter, the solution was added to CHO cells that hadbeen cultured to a high density in a 25 cm² flask containing 5 ml ofHam's F-12 medium (containing 5% FCS). After incubating at 37° C. for 4hours in the presence of 5% CO₂, the medium was replaced with a fleshmedium, followed by subsequent incubation at 37° C. for 20 hours in thepresence of 5% CO₂. Next, the medium was replaced with Ham's F-12 mediumcontaining 5% FCS, 0.4 mg/ml Geneticin (GIBCO BRL Co.), and 10 daysculture was subsequently conducted. In the method, the medium wasreplaced once.

Through this selection by a drug for 10 days, only the transformed cellscould survive and proliferated, however to the contrary, cells that werenot transformed died. In order to obtain highly expressing cells fromthe resulting transformed cells, sorting was performed by a cell sorter(Becton Dickinson Co.). Staining of hSRCL-P1 expressed on the cellsurface was first conducted. After washing the transformed cells in the25 cm flask with 5 ml PBS(−) twice, the cells were unstuck with 0.3 mlof 0.02% EDTA solution (Nakarai Tesc KK). The cells were suspended in 10ml PBS (−), and thereafter centrifuged at 200×g for 7 minutes at 4° C.to remove the supernatant. To the remaining cells, added 50 μl of asolution of an anti-myc antibody (Invitorogen Co.) that was diluted with2% FCS/PBS (−) by ten folds. After intimately suspending the cells, thesuspension was incubated at 4° C. for 20 minutes. Thereafter, 10 ml of2% FCS/PBS (−) was added thereto and suspended, followed by washesthrough the centrifugation at 200×g for 7 minutes at 4° C. and theremoval of the supernatant. To the remaining cells, added 50 μl of asolution of a secondary antibody Alexa488 labeled anti-mouse IgG (H+L)that was diluted with 2% FCS/PBS (−) by ten folds. After intimatelysuspending the cells, the suspension was incubated at 4° C. for 20minutes. Thereafter, 10 ml of 2% FCS/PBS (−) was added thereto andsuspended, followed by washes through the centrifugation at 200×g for 7minutes at 4° C. and the removal of the supernatant. The remaining cellswere suspended in 0.5 ml of 2% FCS/PBS (−) to give a sorting sample.After the sample was passed through a 5 ml tube equipped with a cellstrainer cap (Becton Dickinson Co.), it was applied to a cell sorter.CHO cells without subjecting to the transformation, which had beensimilarly treated, were used as control cells. Accordingly, a sample wasselected, which exhibits fluorescence intensity of 10 times or greaterthan the control sample.

These cells were dispensed into 96-well cell culture plates, of whichwells respectively contained 100 μl Ham's F-12 medium (containing 5%FCS, 0.4 mg/ml Geneticin), to charge a single cell per well. After thecells were cultured at 37° C. in the presence of 5% CO₂ for one week,additionally each 100 μl of a culture medium was added thereto followedby the additional culture for one week. A clone proliferated in the drugselection with Geneticin was divided into two parts, which weresubjected to passages on 12-well and 24-well cell culture plates. Uponthe passage, clones that proliferated from two cells or more per wellwere excluded, and the cells were plated at a cell number ratio of 9: 1to 12-well and 24-well cell culture plates. The cells were cultured at37° C. in the presence of 5% CO₂ until the cells in the 12-well platereach to high density. Then, the cells were stained again similarly tothe procedure where individual clones were subjected to sorting, andthereafter, they were applied to FACSCalibur (Becton Dickinson Co.) todetermine the expression level. After determining a clone exhibiting theexpression at a higher amount, respectively corresponding cells in the24-well plate clone were identified as a stable expression cell strain(CHO/hSRCL-P1).

Example 10 Binding Specificity of hSRCL-P1

Binding specificity of hSRCL-P1 was examined using the stable expressioncell strain CHO/hSRCL-P1, which was obtained in Example 9, for (1) yeast(Zymosan A Bioparticles, manufactured by Molecular Probes Co.); gramnegative bacterium (Escherichia coli Bioparticles, manufactured byMolecular Probes Co.); or gram positive bacterium (Staphylococcus aureusBioparticles, manufactured by Molecular Probes Co.), (2) oxidized LDL(2.0 mg/ml LDL added with 50 μM CuSO₄ followed by subjecting to thereaction for 24 hours and to the dialysis in PBS(−)), (3) AGE-HSA(AGE-human serum albumin, which was prepared according to Ikeda, K. etal., Biochemistry 35(24), 8075-8083 (1996)), or (4) mannose (α-D-MannoseBP-Probe, manufactured by Seikagaku Kogyo KK) or fucose (α-L-FucoseBP-Probe, manufactured by Seikagaku Kogyo KK).

CHO/hSRCL-P1 was first plated on a 35 mm bottom dish (Matsunami glassKK) at 1×10⁵ cells, and cultured at 37° C. for 3 days in the presence of5% CO₂. The culture was conducted in 2 ml of Ham's F-12 mediumcontaining 5% FCS, 0.4 mg/ml Geneticin. After 3 days passed, the cellswere washed twice with 1 ml of Minimum Essential Medium Alpha Mediumcontaining 2% FCS (αMEM/2% FCS). Thereafter, each 1 ml of αMEM/2% FCScontaining 25 μg/ml yeast, 25 μg/ml gram negative bacterium, 25 μg/mlgram positive bacterium, 5 μg/ml oxidized LDL, 10 μg/ml AGE, or 10 μg/mlmannose or 10 μg/ml fucose was added thereto. Three hours reaction at 4°C. was allowed, and thereafter, the cells were washed five times with 1ml of αMEM/2% FCS.

The binding was confirmed as below. First, in regard to (2), (3) and(4), each 1 ml of 100 folds dilution of (2) an anti-oxidizedphosphatidylcholine antibody; (3) an anti-HSA antibody (BIOSYS Co.); or(4) streptavidin, Alexa594 conjugate (Molecular Probes Co.) in αMEM/2%FCS was added, followed by further 30 minutes incubation at 4° C.Thereafter, the cells were washed three times with 1 ml of αMEM/2% FCS.Next, 0.2 ml of a solution of 4% paraformaldehyde/PBS (−) was added toany one of the above (1) to (4), and then the fixation was allowed bythe incubation at room temperature for 20 minutes. Then 1 ml of TBSC(Takara Shuzo Co., Ltd, a buffer containing TBS (Tris-Buffered Saline)Powder which was adjusted to give a predetermined amount with sterilizedand distilled water, and added with CaCl₂ at a final concentration of 5mM) was used for three times washes. Next, in regard to (2) and (3), thereaction with secondary antibody was performed. More specifically, each1 ml of 200 folds dilution of (2) rhodamine labeled anti-mouse IgM MuChain (Chemicon International Co.); or (3) Alexa 594 anti-goat IgG (H+L)(Molecular Probes Co.) in 25% BlockAce (Dainippon Pharmaceutical Co.,Ltd.)/TBSC was added thereto, followed by further 30 minutes incubationat room temperature. Thereafter, the cells were washed three times with1 ml of TBSC. Next, in regard to from (1) to (4), SlowFade LightAntifade Kit (Molecular Probes Co.) was used for the mounting to givesamples for the observation under a fluorescence microscope. For each ofthe samples, the fluorescent image was observed by a fluorescenceobservation system of an inverted system microscope IX70 manufactured byOlympus Co., Ltd. The results are respectively depicted in FIG. 5 for(1) (A: yeast, B: gram negative bacterium (Escherichia coli), and C:gram positive bacterium (Staphylococcus aureus)), and in FIG. 6 for(2)-(4) (A: oxidized LDL, B: mannose, and C: AGE). As is clear fromthese figures, specific binding images could be observed in CHO cellsthat are stably expressing hSRCL-P1 in all cases of from (1) to (4). Theresults shown in FIG. 5, A-C, in which bacterium was employed, clearlyindicated that each of the stained parts of hSRCL-P1 (in each leftfigure, stained in green) overlapped with each of the parts where thebacteria are present (in each middle figure, stained in red), which wasfound as Overlap in each right figure, and that each of the bacteriaspecifically bound to hSRCL-P1.

In addition, cells in which hSRCL-P1 was transiently expressed (see,Example 7) gave the similar results, which demonstrate the specificbinding.

Example 11 Intracellular Incorporation of Binding Complex byPhagocytosis of hSRCL-P1

Intracellular incorporation of each binding complex, which was employedin Example 10, was observed using the transient expression cell andstable expression cell strains of hSRCL-P1 obtained in Examples 7 and 9.Incorporation of the binding complex was confirmed by modifying methoddescribed in Example 10, in which the reaction with the binding complexwas performed at a temperature of 37° C. After staining, theincorporation status within the cells was observed by three-dimensionalimage processing using a confocal laser scanning microscope manufacturedby Olympus Co., Ltd. The results in the examination where the transientexpression cells were used are depicted in FIG. 7 in regard to thoseobtained for yeast, which reveal that yeast cells (stained in red) wereincorporated into cells that were expressing hSRCL-P1 (stained ingreen). Furthermore, similar results were also obtained when the stableexpression cell strain was employed.

Example 12 Demonstration of SRCL-P1 Expression in Vascular EndothelialCells

In order to verify the expression and localization of hSRCL-P1 intissues, fluorescent immunostaining was performed in accordance with themanipulation below using paraffin embedded sections derived from healthyhuman and mouse heart (Novagen Co.).

Slides with the paraffin embedded section was immersed 3 times in xyleneat room temperature for 10 minutes in a stain tray to effect theparaffin removal treatment. Thereafter, the slides were sequentiallyimmersed in 100%-90%-80%-70%-ethanol at room temperature for 10 minuteseach, and into PBS (−) solution for 10 minutes for achieving thehydration treatment.

Next, the slides were immersed in a solution of PBS (−) containing 3%hydrogen peroxide in order to suppress the peroxidase activity thatintrinsically exists on the tissue section at room temperature for 10minutes. Thereafter, blocking was carried out by immersing the slide inBlocking Ace (Dainippon Pharmaceutical Co., Ltd.) at room temperaturefor 1 hour.

Next, 100 μl of an anti-hSRCL-P1 rabbit polyclonal antibody (IgGfraction, 100 μg/ml) as a primary antibody was applied on the tissuesection in a humid box, and subjected to a reaction for 30 minutes. Theprimary antibody washed three times by immersing into a washing solution(Tris-HCl: pH 7.5, 0.15 M NaCl, 0.05% Tween 20) in a staining tray whileshaking for 10 minutes at room temperature. Thereafter, POD (peroxidase)labeled anti-rabbit IgG sheep antibody (Boeringer Mannheim Co., Ltd.) asa secondary antibody was reacted in a similar manner to the primaryantibody at a concentration of 5 U/ml, followed by washes. Then,Biotinyl Tyramide Amplification Reagent (NEN (trade name), manufacturedby Life Science Products Co.) was applied to the slide, and the reactionwas allowed at room temperature for 10 minutes, followed by similarwashes to the procedure for the primary antibody. Avidin Alexa Fluor(trade name) 488 conjugate (manufactured by Molecular Probes Co.) of 1mg/ml was diluted to 100-folds in a solution of PBS (−), and 100 μl ofthe resulting solution was applied to the tissue section on the slide inthe humid box at room temperature, and subjected to a reaction for 30minutes, followed by similar washes to the procedure for the primaryantibody. Thereafter, Slow Fade Light Antifade Kit (Molecular ProbesCO.) was used for the mounting to give a sample for the observation witha fluorescence microscope (Nikon Co.). In addition, a slide for thenegative control was prepared in a similar manner except that normalrabbit serum was used for the reaction instead of the primary antibody.

Consequently, stained images were observed in heart vascular endothelialcells for both of A: healthy human and B: mouse as shown in FIG. 8 (leftfigure each), while such stained images were not found whatever in thenegative control (right figure each). Accordingly, it was verified thatSRCL-P1 was expressed in vascular endothelial cells in the heart,suggesting that SRCL-P1 participates in the binding of oxidized LDL, AGEand the like onto the blood vessel wall.

EFFECT OF THE INVENTION

Because SRCL-P1 protein of the present invention has an SR structure anda collectin structure, it is believed to be a substance that exertscharacteristic effects to those structures. Therefore, it can beutilized in the elucidation of mechanisms of macrophage and basicimmunity; in the elucidation of mechanisms of the development of a widevariety of diseases such as arteriosclerosis, diabetic complications andAlzheimer's disease, hyper β-lipoproteinemia, hypercholesterolemia,hypertriglyceridemia, hypo α-lipoproteinemia, transplantation,atherectomy, post angiogenic restenosis, bacterial infections; in thediagnostic, prophylactic and therapeutic methods thereof; and in thedevelopment of reagents and drugs for the same.

1-32. (canceled)
 33. An isolated and purified polypeptide having anamino acid sequence that comprises amino acid residues 1 to 618 of SEQID NO:
 24. 34. An isolated and purified polypeptide according to claim33, wherein the amino acid sequence of the polypeptide consists of aminoacid residues 1 to 618 of SEQ ID NO:
 24. 35. A method for producing thepolypeptide according to claim 33 comprising: culturing in a medium acell transformed with a polynucleotide that comprises a nucleotidesequence encoding the polypeptide according to claim 33 and collectingthe polypeptide produced by the cells.
 36. The method according to claim35, wherein said cell is of Escherichia coli, an animal or an insect.37. The method according to claim 35, wherein said polynucleotide is aninsert in a vector, and wherein the cell is transformed with the vector.38. A method for producing a monoclonal antibody having specificity forthe polypeptide according to claim 33, comprising: (a) administering thepolypeptide according to claim 33 to an animal, (b) selecting the animalthat exhibits an antibody titer, (c) collecting a spleen or a lymph nodefrom the animal, (d) fusing antibody-producing cells contained thereinwith myeloma cells, and (e) selecting a hybridoma cell line thatsecretes a monoclonal antibody having specificity for the polypeptide ofclaim
 33. 39. A monoclonal antibody produced by the method according toclaim
 38. 40. A monoclonal antibody having specificity for a proteinthat has an amino acid sequence consisting of residues 1 to 618 of SEQ.10 NO:
 24. 41. A method for quantitatively determining the polypeptideaccording to claim 33, comprising: detecting an immunological bindingbetween the polypeptide according to claim 33 and a monoclonal antibodyhaving specificity for said polypeptide; quantifying an amount of thepolypeptide based on the detected immunological binding.
 42. A method ofscreening for a drug for treating a pathological state involved in theaccumulation of oxidized Low Density Lipoprotein (LDL), wherein themethod comprises: measuring binding between the protein according toclaim 33 and oxidized LDL in the presence and absence of a candidatedrug, and identifying a drug for the treatment of a pathological stateinvolved in the accumulation of oxidized LDL by comparing the bindingbetween the protein according to claim 33 and oxidized LDL in thepresence and absence of a candidate drug, wherein an inhibitory abilityof the candidate drug toward the binding between the protein accordingto claim 33 and oxidized LDL identifies the candidate drug as a drug forthe treatment of the pathological state.
 43. A method of screening for adrug for treating a pathological state involved in the binding ofadvanced glycation end products (AGE) to cells, comprising: measuringbinding between the protein according to claim 33 and AGE in thepresence and absence of a candidate drug, and identifying a drug for thetreatment of a pathological state involved in the binding of AGE tocells by comparing the binding in the presence and absence of acandidate drug, wherein an inhibitory ability of the candidate drugtoward the binding between the protein according to claim 33 and AGEidentifies the candidate drug as a drug for the treatment of thepathological state.